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Computer Modeling of Spin Labels
CHAPTER TWENTY-ONE
Computer Modeling of Spin Labels: NASNOX, PRONOX, and ALLNOX Kathleen N. Beasley*,1, Brian T. Sutch*,1, Ma'mon M. Hatmal†, Ralf Langen{, Peter Z. Qin}, Ian S. Haworth*,{,2 *Department of Pharmacology & Pharmaceutical Sciences, University of Southern California, Los Angeles, California, USA † Department of Laboratory Medical Sciences, Faculty of Allied Health Sciences, Hashemite University, Zarqa, Jordan { Department of Biochemistry, University of Southern California, Los Angeles, California, USA } Department of Chemistry and Department of Biological Sciences, University of Southern California, Los Angeles, California, USA 1 These authors contributed equally to the work. 2 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. NASNOX 2.1 Protocol 3. PRONOX 3.1 Protocol 4. ALLNOX 4.1 Standard Label–Linker–Target Assemblies 4.2 Inclusion of a Custom Linker 4.3 Inclusion of a Custom Label and a Custom Target 4.4 Alternative Approaches for Complex Labels 4.5 Further Modifications of the Program References
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Abstract Measurement of distances between spin labels using electron paramagnetic resonance with the double electron–electron resonance (DEER) technique is an important method for evaluation of biomolecular structures. Computation of interlabel distances is of value for experimental planning, validation of known structures using DEER-measured distances, and determination of theoretical data for comparison with experiment. This requires steps of building labels at two defined sites on proteins, DNA or RNA; calculation of allowable label conformers based on rotation around torsional angles; computation of pairwise interlabel distances on a per conformer basis; and calculation of an average distance between the two label ensembles. We have described and validated two programs for this purpose: NASNOX, which permits computation of distances Methods in Enzymology, Volume 563 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2015.07.021
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2015 Elsevier Inc. All rights reserved.
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between R5 labels on DNA or RNA; and PRONOX, which similarly computes distances between R1 labels on proteins. However, these programs are limited to a specific single label and single target types. Therefore, we have developed a program, which we refer to as ALLNOX (Addition of Labels and Linkers), which permits addition of any label to any site on any target. The main principle in the program is to break the molecular system into a “label,” a “linker,” and a “target.” The user can upload a “label” with any chemistry, define a “linker” based on a SMILES-like string, and then define the “target” site. The flexibility of ALLNOX facilitates theoretical evaluation of labels prior to synthesis and accommodates evaluation of experimental data in biochemical complexes containing multiple molecular types.
1. INTRODUCTION Introduction of nitroxide radicals as spin labels at specific sites of biological macromolecules can provide information on molecular structure, flexibility, and environment using electron paramagnetic resonance (EPR). Several recent reviews have described techniques for site-directed spin labeling (Fielding, Concilio, Heaven, & Hollas, 2014; Hubbell, Lo´pez, Altenbach, & Yang, 2013; Jeschke, 2013) and subsequent analyses using spin-labeled proteins (Bordignon & Polyhach, 2013; Klare, 2013; Sahu, McCarrick, & Lorigan, 2013), DNA (Ding et al., 2014), RNA (Esquiaqui, Sherman, Ye, & Fanucci, 2014), and lipids (Gaffney, 2014; Schwarzmann, Arenz, & Sandhoff, 2014). Labeling of a target simultaneously at two sites permits determination of an interspin label distance using the double electron–electron resonance (DEER) method, as described in many of these reviews. The nitroxide radical of the spin label is commonly stabilized by its inclusion as a substituent of a multimethylated ring. The label is connected to the target molecule directly or via a flexible or rigid linker, with a wide variety of linker chemistries used to form the label–linker–target assembly. Some common examples are shown in Table 1, in which we use the “label– linker–target” nomenclature for a specific purpose, as described further below. The R1 and R5 assemblies (which include the same “label”: 1-oxyl-2,2,5,5-tetramethylpyrroline) are the most commonly used for proteins and DNA, respectively, with numerous examples in the literature (see above-referenced reviews). TOAC (a label containing a 1-oxyl-2,2,6,6tetramethylpiperidine ring) is increasingly commonly used as a rigid label in which the Cα of the protein backbone is also part of the label ring (Table 1). TOAC can be incorporated into short peptides for DEER measurements to probe peptide conformation (Milov, Tsvetkov, et al., 2014;
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Table 1 Examples of Spin Labels, Linkers, and Targets Linker Target Labela
a
Common Namesb
CSSC
Protein Cα
R1, MTSL, MTSSL
CS
DNA P
R5
CC
Thymine C5
5sp
None
Protein Cα
TOAC
None
Cytosine N4 Adenine N6
TEMPO
Cytosine N4 Adenine N6
TEMPO
Protein Cα
TOPP
L, linker; T, target. Short-hand names used in the literature to describe the label–linker–target assembly.
b
Sahu et al., 2014; Shabestari et al., 2014) and behavior at organic surfaces (Milov, Samoilova, et al., 2014). The piperidine ring in TOAC may also be used as a DNA label, as recently illustrated by Gophane and Sigurdsson (2015). In this context, the label is referred to as TEMPO and is connected directly or via a linker to a base target (amino group in cytosine or adenine) (Table 1). We return to this label–linker–target assembly in Section 4.3. Spin labels with a variety of
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structures have also been attached to molecules such as cyclodextrin (Krumkacheva et al., 2013), cholesterol (Williams, Wassall, Kemple, & Wassall, 2013), and stearic acid (Liu, Zhang, Fan, Wang, & Jiao, 2013; Reichenwallner & Hinderberger, 2013). The choice of labels may also depend on the experimental conditions, as discussed in a study of in-cell EPR using spin-labeled DNA quadruplexes (Holder, Drescher, & Hartig, 2013). In vivo considerations may become increasingly important, since EPR using spin-labeled stearic acid has been proposed as a diagnostic modality for colorectal cancer (Liu et al., 2013). In theoretical calculations of interspin label distances, accurate modeling of the label conformer distribution is necessary for labels connected to a target with a flexible linker. Three programs, MMM (Polyhach, Bordignon, & Jeschke, 2010), PRONOX (Hatmal et al., 2012), and MtsslWizard (Hagelueken, Ward, Naismith, & Schiemann, 2012) have been developed for computation of distributions of R1 on proteins (Table 1). These programs use distinct, but somewhat similar, approaches to calculation of rotamers in the R1 linker (Fig. 1A), based on information obtained from X-ray data (Guo, Cascio, Hideg, & Hubbell, 2008; Langen, Oh, Cascio, & Hubbell, 2000) and supported by quantum mechanical calculations (Warshaviak, Serbulea, Houk, & Hubbell, 2011). The programs give relatively similar results for interlabel distances based on a detailed evaluation by Jeschke (2013), with each having an RMSD of about 3 A˚ compared to experimental DEER distances. An accurate rotamer library is also available in the RosettaEPR package (Alexander et al., 2013). A direct comparison of theoretically generated label conformers with those obtained biophysically has also recently been described (Florin, Schiemann, & Hagelueken, 2014).
Figure 1 Definitions of rotatable torsional angles in the linkers of (A) the R1 protein label and (B) the R5 DNA label.
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In our development of PRONOX, we validated the program against a large database of experimental DEER distances obtained from R1-labeled proteins (Hatmal et al., 2012). We had previously developed a separate program, NASNOX, for similar use with R5-labeled DNA or RNA, and we have also tested NASNOX using experimental data (Price, Sutch, Cai, Qin, & Haworth, 2007). Both programs compute label distributions via torsional angle rotation in the linker joining the label to the protein or DNA backbone (Fig. 1), with subsequent calculation of all pairwise interconformer distances between the two label distributions and determination of an average interlabel distance. We typically quote this distance as that between the N atoms of the nitroxide groups (Hatmal et al., 2012; Price et al., 2007). The restriction of NASNOX and PRONOX to a single label type at a distinct biomolecular site is advantageous, in that it allows system-specific considerations to be implemented in the algorithms. Such considerations include label–biomolecule interactions and calculation of interlabel distances after weighting favorable conformers in the distribution, as described in Sections 2 and 3. However, the spin labeling field is expanding through use of multiple label types attached to various sites on proteins, DNA/ RNA, and other targets, using a variety of linkers through a range of chemistry. To facilitate calculations on these labels and to allow new labels to be examined theoretically prior to synthesis, we have developed a new program, entitled ALLNOX (Addition of Labels and Linkers). In this chapter, we first briefly describe use of NASNOX and PRONOX, but we mainly refer the reader to previous descriptions of these programs (Hatmal et al., 2012; Price et al., 2007; Qin et al., 2007). We then focus on utilization of ALLNOX for flexible building of spin-labeled biomolecules, automated calculation of label conformer distributions, and computation of interlabel distances. NASNOX, PRONOX, and ALLNOX are available at the URLs shown in Table 2.
Table 2 URLs for NASNOX, PRONOX, and ALLNOXa Program URL
NASNOX
http://pzqin.usc.edu/NASNOX/
PRONOX
https://ihlab.hsc.usc.edu/pronox/
ALLNOX
https://ihlab.hsc.usc.edu/allnox/
a
All the programs can be accessed from the “Programs” dropdown menu at https://ihlab.hsc.usc.edu/.
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2. NASNOX NASNOX is designed to model the conformational distribution of R5 at a DNA or RNA target site (Price et al., 2007; Qin et al., 2007). With the nucleic acid coordinates fixed, the program systematically varies torsion angles t1, t2, and t3 (Fig. 1B) and identifies allowed conformers that have no steric clash between the label and the target molecule. A clash is defined as a contact distance <75% of the sum of the van der Waals radii of the interacting atoms. Interspin label distances are calculated for every pairwise combination of labels in the allowed ensemble at the two sites, and the mean and standard deviation of the distances are then determined. Each R5 conformer distribution and the interspin label distances can be computed in less than a minute on a PC using the default settings. NASNOX uses the R5 label with a CS linker (Table 1) that connects to the DNA or RNA through formation of a phosphorothioate at the target site. An important feature of NASNOX is that a “site” can comprise two label distributions to allow for calculation of distances using spin labeling that result in a racemic mixture of labels at a given phosphodiester. A screenshot of the NASNOX site and a typical output structure are shown in Fig. 2. In this example, label conformer distributions (shown in green (light gray in the print version) and red (dark gray in the print version)) are calculated for labels with sulfur replacing each nonbridging oxygen atom of the phosphodiester at deoxynucleotide 18, and similarly at 36. The calculated interlabel distance is determined from the racemic mixture of labels at both sites. A single enantiomer distribution can also be calculated at a target site. We have described the use of NASNOX elsewhere (Qin et al., 2007) and the reader is directed to this description for further details. The following protocol is given in brief and with the goal of highlighting differences between NASNOX and ALLNOX.
2.1 Protocol Step 1 NASNOX is available as a Web application (http://pzqin.usc.edu/ NASNOX/). The initial screen does not contain default settings, so all numbers need to be entered by the user. Step 2 A target file in pdb format containing DNA or RNA must be uploaded using the “Choose File” button. Any other molecular types in the file will be ignored. The default in NASNOX is to add protons to the DNA or RNA if not already present. The
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Figure 2 Depiction of the NASNOX Web site and a structure resulting from a NASNOX calculation. The green (light gray in the print version) and red (dark gray in the print version) label distributions at each site (18 and 36) are for labels with sulfur replacing the respective nonbridging oxygen atoms at each phosphodiester.
two sites for labeling are entered in the corresponding boxes (Fig. 2: sites 18 and 36 in the example). If labeling at both nonbridging oxygen atoms (replaced by sulfur) is required, then each box “O1P” and “O2P” should be set to “1.” If a single enantiomer label distribution is required at, for example, O1P, then set the “O2P” box to “0.” Note 2.1 The identities of “O1P” and “O2P” in the pdb file are critical. The user should check this carefully, first to ensure that the atoms are named as such, and then to ensure that the correct enantiomer is being chosen. Note 2.2 NASNOX renumbers the input molecule; therefore, in this example, 18 and 36 are the 18th and 36th deoxynucleotides in the DNA. Step 3 The screenshot in Fig. 2 shows the default settings recommended in Price et al. (2007). Torsions t1 and t2 can adopt gauche(+), trans, and gauche() (60°, 180°, and 300°) positions (starting value 60°, three steps), and torsion t3 is varied from 0° to 330° in steps of 30° (starting value 30°, 12 steps).
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Note 3.1 The “Hydrophobic Contacts” option is unlikely to alter the results significantly, but this option might be useful if the label is likely to come into contact with a thymine methyl group. Note 3.2 The “Fine Search” option is unlikely to be needed for calculations on duplexes and other structures in which the label is not spatially limited. This option is useful in situations in which an initial run fails to locate an acceptable label conformer. The “Fine Search” option can be switched on for each torsion separately by setting the corresponding box to “1.” This option searches around the initial torsional setting (Price et al., 2007) to locate acceptable conformers. Using this option for all torsion angles will substantially increase the running time. Step 4 Click the “Submit” button. The results will appear on a new screen within a few seconds. A structure file (data001lig.pdb) and a text results file (data.add) can be downloaded. The structure file contains all the allowable label conformers, listed in order of site after the DNA or RNA coordinates. The results file includes details of the fate of each conformer (allowed or not allowed) and each pairwise distance (shown for N–N, O–O, and midpoint NO(mNO)–mNO distances) between allowed conformers at the two sites. The average N–N, O–O, and mNO–mNO distances and standard deviations are given at the end of the results file.
3. PRONOX PRONOX was developed for computation of interlabel distances between two R1-labeled protein sites (Table 1), based on calculation of spin label conformer distributions through variation of torsions χ1 to χ5 (Fig. 1A; Hatmal et al., 2012). Each label is added using the Cβ atom of the original amino acid (or guided by Hα for glycine). The geometry of the CSSC linker is based on crystallographic data (Langen et al., 2000). The nitroxide ring geometries are described in Hatmal et al. (2012), with reference to earlier work in Price et al. (2007). Conformers generated by rotation around χ1 to χ5 are accepted if they do not clash with the protein. A clash is defined as a label atom to protein atom distance of <75% of the sum of the van der Waals radii of the two atoms. This percentage is
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adjustable in the input and adjustment may be necessary for labels that are located in spatially restricted locations. An important aspect of PRONOX is the weighting of conformers that are known to be favorable based on X-ray evidence showing that the χ1 and χ2 torsions are well defined (Guo et al., 2008). The χ1, χ2 gauche(), gauche() (m,m); trans, gauche(+) (t,p); and trans, gauche() (t,m) conformers have a nonbonded interaction between Sδ and Hα ((m,m) and (t,p)) or other backbone atoms ((t,m)) and this makes these conformations favorable. Thus, the default in PRONOX is a mode in which conformers with these values for χ1 and χ2 are included in the distribution with a biased weighting (Hatmal et al., 2012). In the default setting, the torsional angle around the S–S bond (χ3) is taken to be 90° and all positions of χ4 and χ5 are considered to be isoenergetic. A screenshot of the PRONOX Web site and a typical output structure is shown in Fig. 3.
3.1 Protocol Step 1 PRONOX is available as a Web application (https://ihlab.hsc.usc. edu/pronox/). The initial screen is populated with all default settings. For most runs, all the user needs to enter are the two amino acid residue numbers (11 and 21 in the example in Fig. 3).
Figure 3 Depiction of the PRONOX Web application and a structure resulting from a PRONOX calculation. The R5 distributions are shown in orange (dark gray in the print version) and blue (dark gray in the print version) at sites 11 and 21, respectively.
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Step 2 A file in pdb format containing a protein should be uploaded. Any nonprotein atoms will be ignored. The default in PRONOX is not to add protons to the protein. Note 2.1 PRONOX renumbers the input protein; therefore, 11 and 21 are the 11th and 21st amino acids in the pdb input file. Step 3 The default settings for PRONOX should be satisfactory for labels that are not in a spatially restricted position. Torsions are defined using a starting value and a step number: for example, a starting value of 60° and a step of three gives torsional values of 60°, 180°, and 300°. Note 3.1 If the label is spatially restricted, the “vdW” setting can be modified to alter the clash criterion. We have found that a setting of 0.40 (the “low clash” mode) is effective for hindered labels (Hatmal et al., 2012). Note 3.2 The “Fine Search” option (as described for NASNOX) is also available for each torsion angle (by adding a check mark to the appropriate box) for labels in restricted locations. Note 3.3 For a spatially restricted label, the torsion increments can also be decreased, but we do not recommend this approach since it is not chemically justifiable. Step 4 We recommend that the default weighting is maintained in all runs. Note 4.1 Setting the “Weight” box to 0.5 will result in all labels being weighted equally. Step 5 If the target amino acid is a glycine, the “Glycine” box must be checked. All other boxes can be modified as in Steps 2–4. For addition of a label to glycine, the pdb file must contain a proton on the glycine Cα, and the proton must be named “HA3.” It is important to ensure that the correct prochiral proton is identified and named “HA3” in the pdb file. Step 6 Click the “Run PRONOX” button. The results will appear on a new screen within a few seconds. A structure file (output.pdb) and a text results file (data.add) can be downloaded. Note that these files will have a random computer-generated string in the file name after download. The structure file contains all the allowable label conformers, listed in order immediately after the amino acid of each labeled site in the pdb file. The results file includes details of each conformer (allowed or not allowed, favorable or unfavorable for
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weighting). After this listing for each site, the total number of favorable and unfavorable labels identified for the site is given, with a % in parentheses indicating how much each of these labels contributes to the conformer distribution. Each pairwise distance (N–N, O–O, and midpoint NO(mNO)–mNO) is shown between allowed conformers at the two sites. The average N–N, O–O, and mNO– mNO distances and standard deviations are given at the end of the results file. The total number of interlabel distances in the calculation is given in parentheses. Note 6.1 A useful feature of the PRONOX Web application is that each run has a designated job ID and results can be recovered using this ID. However, only a limited number of results are saved and there is currently no login enabled, so users should generally download results as they are obtained.
4. ALLNOX NASNOX and PRONOX are useful for computation of interlabel distances for R5-labeled DNA or RNA and R1-labeled proteins, respectively. However, these programs are limited in their restriction to a single label type and a single biomolecular target. To overcome this limitation, we have developed an algorithm that we refer to as ALLNOX. This program was written to allow the user to add a spin label with any chemistry to any site on any molecular target, with automated computation of label conformer distributions and calculation of interlabel distances based on these distributions. We begin by defining each labeled site as a “label–linker–target” assembly (Table 1). In this context, the “label” is the ring that contains the nitroxide radical (although this definition is not absolute); the “linker” is the chain of atoms between the label and the target, and is generally the source of conformational flexibility; and the “target” is the biomolecule (protein or DNA in the examples below, but this can be any molecule). In developing the ALLNOX Web application, we have tried to maintain the relative simplicity of the NASNOX and PRONOX Web applications and avoid the requirement for too many options. This is challenging given the need to accommodate user-defined label–linker–target assemblies that are unknown in advance.
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In the following sections, we describe use of ALLNOX for several different situations. In Section 4.1, we start with building of standard label– linker–target assemblies similar to those in NASNOX and PRONOX. In Sections 4.2 and 4.3, we illustrate the assembly design with increasingly more user-defined “unknown” elements.
4.1 Standard Label–Linker–Target Assemblies The standard label–linker–target assemblies that are currently implemented in ALLNOX are listed in Table 3, and are available from a dropdown menu on the ALLNOX Web site. We use common nomenclature to indicate these assemblies; for example, R1 and R5 are used to indicate the 1-oxyl-2,2,5,5-tetramethylpyrroline “label.” Some of the standard assemblies produce label–linker–target arrangements similar to those in NASNOX and PRONOX, but the calculated interlabel distances are not ˚ , which is within exactly the same; however, they are generally within 2 A the error of these calculations ( Jeschke, 2013). This is because in ALLNOX protons are not added to DNA (unlike in NASNOX) and no weighting of conformers is performed (unlike in PRONOX). 4.1.1 Protocol Step 1 ALLNOX is available as a Web application (https://ihlab.hsc.usc. edu/allnox/). The initial screen has input fields for the minimal options required for a run using standard label–linker–target assemblies at both sites. Table 3 Standard Label–Linker–Target Assemblies in ALLNOX Program Definition
R1-CSSC-aaCα
R1-labeled protein (similar to PRONOX)
R1-CSSC-GlyCα
R1-labeled protein at glycine (similar to PRONOX for glycine)
R5-CS-nucP
R5-labeled nucleotide (similar to NASNOX, connected via O1P)
R5-CC-ThyC5
Rigid label attached to thymine (5sp in Table 1)
TOAC(Cα)
TOAC with amino acid Cα in the label ring
TOPP-aaCα
TOPP-labeled protein (see Table 1)
TOPP-GlyCα
TOPP-labeled protein at glycine (see Table 1)
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Step 2 A pdb file containing the target (which can be any type of molecule) should be uploaded (Fig. 4A, red (dark gray in the print version) arrow). Most pdb files from common sources should work without alteration, other than elimination of unwanted chains. The default in ALLNOX is not to add protons. Currently, no protons should be included in the input pdb file. ALLNOX also renumbers input molecules (see Step 2 for NASNOX and PRONOX). Step 3 The next step is to define the label–linker–target assembly. Standard options (Table 3) are available via a dropdown menu for assemblies 1 and 2 (Fig. 4A, orange (light gray in the print version) arrows). In the example shown in Fig. 4A, the following assemblies were selected: “Label-linker-target 1”: “R1-CSSC-aaCα,” giving a PRONOX-like label at a protein site to be defined in Step 5. “Label-linker-target 2”: “R5-CS-nucP,” giving a NASNOXlike label at a nucleotide site to be defined in Step 5. Note 3.1 If the target amino acid is a glycine, “R1-CSSC-GlyCα” should be selected. Note 3.2 “R5-CS-nucP” assumes connection to DNA through replacement of the phosphodiester O1P atom with S. Attachment of labels (including R5) with other connectivity to DNA (including at the phosphodiester) is described in Section 4.3. Note 3.3 Other standard assemblies are discussed in Section 4.4. Step 4 The “vdW” boxes are defaulted to 0.75 as the recommended value. This value can be reduced if no results are produced in an initial run (see Step 3 for PRONOX). Step 5 The target sites are set by entering a residue number in each box (Fig. 4A, green (dark gray in the print version) arrows). This selection should reflect the choice of the label–linker–target assembly, as follows in the example in Fig. 4A: “Target Residue Number” 11 refers to the protein (or proteins) in the target pdb file because the standard label–linker–target (R1-CSSC-aaCα) includes a protein target. “Target Residue Number” 18 refers to the DNA in the target pdb file because the standard label–linker–target (R5-CS-nucP) includes a DNA target.
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Figure 4 Depiction of the input fields on the ALLNOX Web application and a structure output for an ALLNOX run using standard label–linker–target assemblies at both sites. (A) Selections for the target, label–linker–target assemblies, target residue numbers, and conformation are indicated by red (dark gray in the print version), orange (light gray in the print version), green (dark gray in the print version), and blue (dark gray in the print version) arrows, respectively. (B). Table of default linker torsions that can be edited by the user. This table is generated in “Manual” conformation mode. Each torsional variation is defined by a minimum and maximum value and an increment. The four atoms comprising the torsional angle are shown on the right. Atom names in the linker are indicated by “L” as the last character. (C). A structure (output.pdb) resulting from an ALLNOX calculation for a protein–DNA complex, showing the R1 label distribution on amino acid 11 (yellow (light gray in the print version)) and the R5 label distribution on deoxynucleotide 18 (blue (dark gray in the print version)).
Note 5.1 ALLNOX renumbers the input molecule; therefore, in this example, 11 is the 11th amino acid in the protein and 18 is the 18th deoxynucleotide in the DNA. Step 6 The last selection before running the simulation is to choose a conformation mode (Fig. 4A, blue (dark gray in the print version) arrow). The following options are available:
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Static: Static mode will generate a single fixed conformation for each label–linker assembly and will ignore clashes with the target. This mode is useful as an initial check, particularly for a user-defined label–linker–target assembly (see Sections 4.2 and 4.3). Automatic: This is the default mode, in which ALLNOX generates a conformational distribution of the label–linker system. Rotatable torsions (single bonds) are varied as gauche(+) (60°), trans (180°), and gauche() (300°), except for the torsion around an S–S bond (90°, as in PRONOX). Manual: This mode will generate a table of the default linker torsions that can be freely edited by the user (Fig. 4B). Atoms in each label–linker–target assembly are shown at the top of this page. Each torsional variation is defined by a minimum and maximum value and an increment. The four atoms comprising the torsional angle are also shown on the right. Atom names in the linker are written as the character in the input linker string, the position of the atom in the string (1 for the atom closest to the label), and “L” as the last character. Note 6.1 In “Static” or “Automatic” mode, clicking the “Run” button will initiate the ALLNOX calculation of the label–linker–target assemblies and interlabel distances. Note 6.2 In “Manual” mode, clicking the “Run” button will produce the editable table of torsion angles. Once this is edited (or simply checked for correctness), clicking the “Run” button again will initiate the ALLNOX calculation. Note 6.3 In “Manual” mode, a torsion can be fixed by entering the same minimum and maximum value and including any positive number (do not enter 0) as the increment. Step 7 The results will appear on a new screen within a minute. A structure file (“output.pdb,” depicted in Fig. 4C) and a text results file (data. add) will be generated and can be downloaded. These files will have a random computer-generated string in the file name after download. The structure file contains all allowable label conformers, listed after the contents of the target pdb file. The results file includes details for each conformer, the total number of conformations found for each label–linker assembly, the pairwise N–N and O–O (nitroxide group) distances between all allowed conformers at the two sites, and the average N–N and O–O distances at the
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end of the file. The total number of interlabel distances in the calculation is given in parentheses. Note 7.1 Each run has a designated job ID and results can be recovered using this ID. However, only a limited number of results are saved and there is currently no login enabled, so users should generally download results as they are obtained. Note 7.2 User registration and account creation will be available in a forthcoming version of ALLNOX, allowing users to perform advanced calculations and save run data on the server.
4.2 Inclusion of a Custom Linker There are a relatively small number of labels used in spin labeling (although this number is growing) and differences in labeling approaches often lie in linker variation. To accommodate modeling of standard labels linked to standard targets through a “non-standard” (custom) linker, ALLNOX permits the user to enter the linker chemistry in a format that is similar to a limited and slightly altered SMILES (simplified molecular-input line-entry system) format (Weininger, 1988). The current limits on the linker are a maximum of 10 atoms with no branches, including carbon (C or c), oxygen (O), nitrogen (N or n), sulfur (S), and phosphorus (P): upper case C or N indicates an sp3 C atom or an amine N, and lower case c or n indicates sp2 C or amide N. Functional groups and bonds (for which the geometry should be correct) based on these combinations are ether, thioether, amine, amide, alkene, and S–S, O–P, and S–P bonds. The following protocol should be read with additional reference to Section 4.1. 4.2.1 Protocol Step 1 To initiate customization of a label–linker–target assembly, the user chooses “Custom” in the dropdown menu below “Label-LinkerTarget 1” or “Label-Linker-Target 2” (Fig. 5A, red (dark gray in the print version) arrows). This selection generates two new dropdown menus under titles of “Label Selection” (Fig. 5A, orange (light gray in the print version) arrows) and “Target Selection” (Fig. 5A, green (dark gray in the print version) arrows), and a “Linker” box (Fig. 5A, blue (dark gray in the print version) arrow). Step 2 The user may select a standard label or a custom label (see Section 4.3). In the example in Fig. 5A, a R1(R5) standard label
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Figure 5 Depiction of the input fields for the ALLNOX Web application and a structure output for an ALLNOX run using standard labels and targets, and a custom linker at both sites. (A) Selections for the label–linker–target assemblies, labels, targets, and linker boxes are identified by red (dark gray in the print version), orange (light gray in the print version), green (dark gray in the print version), and blue (dark gray in the print version) arrows, respectively. (B) Table of default linker torsions. (C) A structure (output.pdb) resulting from an ALLNOX calculation for a protein–DNA complex, showing the label distributions with custom linker CCSSC on amino acid 11 (green (dark gray in the print version)) and custom linker CncCS on deoxynucleotide 18 (pink (dark gray in the print version)).
is chosen at both sites (Fig. 5, orange (light gray in the print version) arrows). Note 2.1 R1(R5) indicates the 1-oxyl-2,2,5,5-tetramethylpyrroline ring (Table 1). We use the R1(R5) terminology because R1 is typically used to describe this label when connected (via a CSSC linker) to proteins, whereas R5 is used when the same ring is connected (via a CS linker) to DNA or RNA. Step 3 The user may also select a standard target or a custom target (see Section 4.3). In the example in Fig. 5A, an aaCα standard target
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(Cα for any amino acid except glycine) is chosen for label–linker– target assembly 1, and a nucP standard target (nucleotide phosphodiester P) is chosen for label–linker–target assembly 2 (Fig. 5A, green (dark gray in the print version) arrows). Step 4 The custom linker is entered in the “Linker” box for each label– linker–target assembly (Fig. 5A, blue (dark gray in the print version) arrow). In entering the linker sequence, it is important to understand that the left most atom will be attached to the label and the right most atom will be attached to the target. In the example in Fig. 5A, a CCSSC linker is used for label–linker–target assembly 1 (that is, an additional C atom compared to the standard linker in the R1-CSSC-Cα assembly) and a CncCS linker is used for label–linker–target assembly 2 (i.e., inclusion of an additional C and an amide compared to the standard linker in the R5-CS-nucP assembly). Note 4.1 ALLNOX can accommodate linker sequences up to a maximum of 10 heavy atoms with single and double bonds, but currently excluding branches. See above for allowable linkers. Note 4.2 A more complete SMILES format (Weininger, 1988) for entry of the linker atoms will be available in a forthcoming version of ALLNOX. Note 4.3 When running a custom linker sequence with >five atoms, the run may take several minutes to complete. Step 5 The “Manual” mode is recommended if a custom linker is used. This allows the user to check that the torsional angles have been set correctly in the editable table. This table is shown in Fig. 5B for the example used in this protocol. ALLNOX recognizes the amide bond and does not include this as a rotatable torsion. Step 6 The results are presented as described in Step 7 of Section 4.1. The output file for the example described in this section is depicted in Fig. 5C.
4.3 Inclusion of a Custom Label and a Custom Target The ultimate goal of ALLNOX is to allow the user to build a custom “label” connected via a user-defined “linker” to a custom “target” (Fig. 6). This requires the user to upload a structure file for the label and to define the topologies and geometries of the label–linker and linker–target connections.
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This approach is facilitated by additional options on the ALLNOX Web site, as described below. We have tried to minimize the amount of information requested of the user and the current program is capable of understanding the chemistry of many label–linker and linker–target connections. This will be improved as we obtain feedback on the functionality of the program. In this section, we use the example of the TEMPO label–linker–target assembly with an amide linker and a cytosine N4 (amino group) target (Table 1; Fig. 7B). Synthesis of this assembly was recently described by Gophane and Sigurdsson (2015). To include this label in ALLNOX, a pdb file for the TEMPO ring (Table 1) was generated from the following SMILES string: C1C(C)(C)N(¼O)C(C)(C)CC1. This example illustrates how ALLNOX can be used to generate a model of a custom label– linker–target assembly. Understanding of the utilization of the program
Figure 6 Depiction of the input fields for an ALLNOX run using a custom assembly. Selections for the label–linker–target assemblies, labels, targets, label atom numbers, target atom names, label to linker geometries, and linker to target geometries are indicated by red (dark gray in the print version), orange (light gray in the print version), black, green (light gray in the print version), yellow (light gray in the print version), blue (dark gray in the print version), and pink (dark gray in the print version) arrows, respectively. The structure shows the atom names used to define the geometry between the label (red (dark gray in the print version)) (k, j, and i) and linker (green (light gray in the print version)) (h) and between the linker (green (light gray in the print version)) (x, y, and z) and target (blue (dark gray in the print version)) (a, b, and c). Note that atoms “i,” “j,” “k” and “a,” “b,” “c” are shown as bonded atoms, but this is not a requirement.
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Figure 7 (A) Edited torsion angle table for the TEMPO-amide-cytosine N4 label–linker– target assembly. Torsions t1 and t2 are set at 180° for both assemblies (sites 19 and 6). (B) Structure of the label–linker–target assembly showing the positions of torsions t1, t2, and t3. (C) Result of an ALLNOX calculation at site 19, showing three conformers (red (dark gray in the print version), green (light gray in the print version),and blue (dark gray in the print version)) generated for the three positions (60°, 180°, and 300°) for torsion t3.
for the complete user-defined assembly may require initial reading of Sections 4.1 and 4.2. 4.3.1 Protocol Step 1 To initiate custom upload of a pdb file containing a custom label, the user first chooses “Custom” in the dropdown menu below “Label-Linker-Target 1” or “Label-Linker-Target 2” (Fig. 6, red (dark gray in the print version) arrows), and then chooses “Custom” again in the “Label Selection” dropdown menu (Fig. 6, orange (light gray in the print version) arrows). Step 2 The selection in Step 1 reveals a “Choose File” upload button, with which the pdb file for the label can be uploaded (TEMPU_UC.pdb in the example in Fig. 6). Note 2.1 The pdb file for the “label” may be generated from a SMILES string using a Web site such as “SMILES
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Translator” (http://cactus.nci.nih.gov/translate/; settings: “PDB,” “3D,” upload the SMILES string as a .txt file). Note 2.2 The PubChem Web site (https://pubchem.ncbi.nlm. nih.gov/) or commercial sites such as that at Santa Cruz Biotechnology (http://www.scbt.com/chemicalstable-spin_traps_spin_probes_and_spin_labels.html) are useful sources of SMILES strings for spin labels. Note 2.3 Make sure that the nitroxide group in the SMILES string is represented as N¼O. This ensures the correct geometry in the resulting pdb file (with use of NO, the oxygen is interpreted as a hydroxyl group). Note 2.4 Some editing of the pdb file (deletion of atoms, such as protons at the atom connecting to the linker) may be needed to produce the appropriate file for input into ALLNOX. Note 2.5 ALLNOX will recognize the nitroxide group automatically. No specific atom names are required in the pdb file. Step 3 The “Linker” boxes should be completed (see Step 4 in Section 4.2). Step 4 The selection in Step 1 produces “Label Atom Number” boxes (Fig. 6, green (light gray in the print version) arrow) in which atoms “i,” “j,” and “k” in the label can be defined. An illustration depicting the location of these atoms is shown in Fig. 6. The numbers for “i,” “j,” and “k” should correspond to the order of atoms in the label pdb file. Note 4.1 Atoms “i,” “j,” and “k” are any three atoms in the label, with “i” connected to the linker (or to the target, if there is no linker). Atom “h” is the first atom of the linker. Step 5 The selection in Step 1 reveals “Label to Linker Geometry” boxes (Fig. 6, blue (dark gray in the print version) arrow). These allow definition of the bond length (distance) “i–h,” angle “j–i–h,” and torsion “k–j–i–h” (see structure in Fig. 6 for definition of atoms “k,” “j,” “i,” and “h”). ALLNOX will make a best guess at these geometries. The values can be edited by the user. Step 6 In the “Target Selection” boxes (Fig. 6, black arrows), the user can choose a standard target or a custom target. If a standard target is chosen, no further input is required. If a custom target is chosen,
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two new boxes appear: “Target Atom Name” (Fig. 6, yellow (light gray in the print version) arrow) and “Linker to Target Geometry” (Fig. 6, pink (dark gray in the print version) arrow). Step 7 In the “Target Atom Name” boxes (Fig. 6, yellow (light gray in the print version) arrow) the atom names for atoms “a,” “b,” and “c” in the target can be defined. An illustration depicting the location of these atoms is shown in Fig. 6. An atom name should be entered in the “Replacement” box to indicate if an atom within the target is going to be replaced by an atom in the linker (or in the label if there is no linker). This box can be left blank if no target atom is to be replaced (as in the example of the TEMPO-derived spin label). Step 8 The “Linker to Target Geometry” boxes (Fig. 6, pink (dark gray in the print version) arrow) allow definition of the bond length (distance) “z–a,” angle “z–a–b,” torsion “z–a–b–c,” angle “y–z–a,” torsion “y–z–a–b,” and torsion “x–y–z–a” (see structure in Fig. 6 for definition of atoms “x,” “y,” “z,” “a,” “b,” and “c”). ALLNOX will make a best guess at these geometries. The values can be edited by the user. Note 8.1 Atoms “a,” “b,” and “c” are any three atoms in the target, with “a” connected to the linker (or to the label, if there is no linker). Atoms “x,” “y,” and “z” are the last three atoms of the linker (“z” is the last atom). Step 9 The “Manual” conformation mode should be used to check that the correct linker torsion angle variations have been defined and to make changes (see Step 5 of Section 4.2). Step 10 The results are presented as described in Step 7 of Section 4.1. The results of the protocol for the TEMPO-amide-cytosine N4 label– linker–target assembly are shown in Fig. 7. First, we note that this is an example in which the program does not have information on the linker to target geometry of the urea group formed in the assembly (Fig. 7B). Thus, the original torsion table had minimum, maximum, and increment values of 60°, 300°, and 120°, respectively. The torsion table in Fig. 7A is shown after editing of torsions t1 and t2 (for both label–linker–target assemblies) such that these torsions are fixed at 180°. This is an example of an assembly that requires some user intervention, but this is straightforward using the Web site. With this change, ALLNOX produces label conformers at both assembly sites. Three conformers are found at site 19 (Fig. 7C).
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4.4 Alternative Approaches for Complex Labels Some spin labels do not fit into the label–linker–target paradigm used in the protocols described above. However, such labels can be accommodated in ALLNOX. The simplest approach is to include the “linker” as part of the uploaded pdb file for the “label.” For a label such as sp5 (Table 1) this is acceptable since the “linker” is a triple bond that is clearly rigid. For TOPP (Table 1), the 1-oxyl-2,2,6,6-tetramethyl-3,5-dioxopiperazine “label” is connected through an aromatic ring to a Cα carbon. This label can be included as part of a standard label–linker–target assembly in the dropdown menu (Table 3), but rotation of the nitroxide-containing ring with respect to the aromatic ring cannot currently be performed. Finally, we note that inclusion of a racemic mixture of R5 (or other labels) at a DNA phosphodiester (which is possible for R5 in NASNOX) is not implemented as a standard label–linker–target assembly in the version of ALLNOX described here. Incorporation of this option is among the planned modifications of the program.
4.5 Further Modifications of the Program We plan further developments of ALLNOX that include increasing the degree of automation of calculation of label conformer distributions; increasing flexibility in linker definition (e.g., including branched linkers); weighting of conformer distributions based on calculation of label–target interactions, target flexibility, and expanding the program to include other targets. One particular area of interest is the growing number of bifunctional spin labels, as illustrated by bisMTSL ( Jeschke, 2013) and the novel DNAtargeted R5c label (Nguyen, Popova, Hideg, & Qin, 2015), both of which have two linker arms. There is potential for use of ALLNOX in experimental design using these labels, based on computation of label conformers through variation of one arm of the label combined with a search algorithm to find possible reactive positions for the second arm. The methods described in this chapter for nitroxide spin labels are also applicable to other spin labels, such as chelated Gd3+ ions (Goldfarb, 2014) and trityl labels (Reginsson, Kunjir, Sigurdsson, & Schiemann, 2012; Shevelev et al., 2014; Yang et al., 2012).
REFERENCES Alexander, N. S., Stein, R. A., Koteiche, H. A., Kaufmann, K. W., McHaourab, H. S., & Meiler, J. (2013). RosettaEPR: Rotamer library for spin label structure and dynamics. PLoS One, 8, e72851.
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Bordignon, E., & Polyhach, Y. (2013). EPR techniques to probe insertion and conformation of spin-labeled proteins in lipid bilayers. Methods in Molecular Biology, 974, 329–355. Ding, Y., Nguyen, P., Tangprasertchai, N. S., Reyes, C. V., Zhang, X., & Qin, P. Z. (2014). Nucleic acid structure and dynamics: Perspectives from site-directed spin labeling. Electron Paramagnetic Resonance, 24, 122–147. Esquiaqui, J. M., Sherman, E. M., Ye, J., & Fanucci, G. E. (2014). Site-directed spin-labeling strategies and electron paramagnetic resonance spectroscopy for large riboswitches. Methods in Enzymology, 549, 287–311. Fielding, A. J., Concilio, M. G., Heaven, G., & Hollas, M. A. (2014). New developments in spin labels for pulsed dipolar EPR. Molecules, 19, 16998–17025. Florin, N., Schiemann, O., & Hagelueken, G. (2014). High-resolution crystal structure of spin labelled (T21R1) azurin from Pseudomonas aeruginosa: A challenging structural benchmark for in silico spin labelling algorithms. BMC Structural Biology, 14, 16. Gaffney, B. J. (2014). Connecting lipoxygenase function to structure by electron paramagnetic resonance. Accounts of Chemical Research, 47, 3588–3595. Goldfarb, D. (2014). Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy. Physical Chemistry Chemical Physics, 16, 9685–9699. Gophane, D. B., & Sigurdsson, S. T. (2015). TEMPO-derived spin labels linked to the nucleobases adenine and cytosine for probing local structural perturbations in DNA by EPR spectroscopy. Beilstein Journal of Organic Chemistry, 11, 219–227. Guo, Z., Cascio, D., Hideg, K., & Hubbell, W. L. (2008). Structural determinants of nitroxide motion in spin-labeled proteins: Solvent-exposed sites in helix B of T4 lysozyme. Protein Science, 17, 228–239. Hagelueken, G., Ward, R., Naismith, J. H., & Schiemann, O. (2012). In silico spin-labeling and generation of distance distributions in PyMOL. Applied Magnetic Resonance, 42, 377–391. Hatmal, M. M., Li, Y., Hegde, B. G., Hegde, P. B., Jao, C. C., Langen, R., et al. (2012). Computer modeling of nitroxide spin labels on proteins. Biopolymers, 97, 35–44. Holder, I. T., Drescher, M., & Hartig, J. S. (2013). Structural characterization of quadruplex DNA with in-cell EPR approaches. Bioorganic & Medicinal Chemistry, 21, 6156–6161. Hubbell, W. L., Lo´pez, C. J., Altenbach, C., & Yang, Z. (2013). Technological advances in site-directed spin labeling of proteins. Current Opinion in Structural Biology, 23, 725–733. Jeschke, G. (2013). Conformational dynamics and distribution of nitroxide spin labels. Progress in Nuclear Magnetic Resonance Spectroscopy, 72, 42–60. Klare, J. P. (2013). Site-directed spin labeling EPR spectroscopy in protein research. Biological Chemistry, 394, 1281–1300. Krumkacheva, O. A., Fedin, M. V., Polovyanenko, D. N., Jicsinszky, L., Marque, S. R. A., & Bagryanskaya, E. G. (2013). Structural equilibrium in new nitroxide-capped cyclodextrins: CW and pulse EPR study. The Journal of Physical Chemistry B, 117, 8223–8231. Langen, R., Oh, K. J., Cascio, D., & Hubbell, W. L. (2000). Crystal structures of spin labeled T4 lysozyme mutants: Implications for the interpretation of EPR spectra in terms of structure. Biochemistry, 39, 8396–8405. Liu, Z., Zhang, W., Fan, S., Wang, L., & Jiao, L. (2013). Changes in the electron paramagnetic resonance spectra of albumin-associated spin-labeled stearic acid as a diagnostic parameter of colorectal cancer. World Journal of Surgical Oncology, 11, 223. Milov, A. D., Samoilova, R. I., Tsvetkov, Y. D., Peggion, C., Formaggio, F., & Toniolo, C. (2014). Peptides on the surface. PELDOR data for spin-labeled alamethicin F50/5 analogues on organic sorbent. The Journal of Physical Chemistry B, 118, 7085–7090. Milov, A. D., Tsvetkov, Y. D., Bortolus, M., Maniero, A. L., Gobbo, M., Toniolo, C., et al. (2014). Synthesis and conformational properties of a TOAC doubly spin-labeled analog of the medium-length, membrane active peptaibiotic ampullosporin a as revealed by CD, fluorescence, and EPR spectroscopies. Biopolymers, 102, 40–48.
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Nguyen, P. H., Popova, A. M., Hideg, K., & Qin, P. Z. (2015). A nucleotide-independent cyclic nitroxide label for monitoring segmental motions in nucleic acids. BMC Biophysics, 8, 6. Polyhach, Y., Bordignon, E., & Jeschke, G. (2010). Rotamer libraries of spin labeled cysteines for protein studies. Physical Chemistry Chemical Physics, 13, 2356–2366. Price, E. A., Sutch, B. T., Cai, Q., Qin, P. Z., & Haworth, I. S. (2007). Computation of nitroxide-nitroxide distances in spin-labeled DNA duplexes. Biopolymers, 87, 40–50. Qin, P. Z., Haworth, I. S., Cai, Q., Kusnetzow, A. K., Grant, G. P., Price, E. A., et al. (2007). Measuring nanometer distances in nucleic acids using a sequenceindependent nitroxide probe. Nature Protocols, 2, 2354–2365. Reginsson, G. W., Kunjir, N. C., Sigurdsson, S. T., & Schiemann, O. (2012). Trityl radicals: Spin labels for nanometer-distance measurements. Chemistry, 18, 13580–13584. Reichenwallner, J., & Hinderberger, D. (2013). Using bound fatty acids to disclose the functional structure of serum albumin. Biochimica et Biophysica Acta, 1830, 5382–5393. Sahu, I. D., Hustedt, E. J., Ghimire, H., Inbaraj, J. J., McCarrick, R. M., & Lorigan, G. A. (2014). CW dipolar broadening EPR spectroscopy and mechanically aligned bilayers used to measure distance and relative orientation between two TOAC spin labels on an antimicrobial peptide. Journal of Magnetic Resonance, 249C, 72–79. Sahu, I. D., McCarrick, R. M., & Lorigan, G. A. (2013). Use of electron paramagnetic resonance to solve biochemical problems. Biochemistry, 52, 5967–5984. Schwarzmann, G., Arenz, C., & Sandhoff, K. (2014). Labeled chemical biology tools for investigating sphingolipid metabolism, trafficking and interaction with lipids and proteins. Biochimica et Biophysica Acta, 1841, 1161–1173. Shabestari, M. H., van Son, M., Moretto, A., Crisma, M., Toniolo, C., & Huber, M. (2014). Conformation and EPR characterization of rigid, 310-helical peptides with TOAC spin labels: Models for short distances. Biopolymers, 102, 244–251. Shevelev, G. Y., Krumkacheva, O. A., Lomzov, A. A., Kuzhelev, A. A., Rogozhnikova, O. Y., Trukhin, D. V., et al. (2014). Physiological-temperature distance measurement in nucleic acid using triarylmethyl-based spin labels and pulsed dipolar EPR spectroscopy. Journal of the American Chemical Society, 136, 9874–9877. Warshaviak, D. T., Serbulea, L., Houk, K. N., & Hubbell, W. L. (2011). Conformational analysis of a nitroxide side chain in an α-helix with density functional theory. The Journal of Physical Chemistry B, 115, 397–405. Weininger, D. (1988). SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. Journal of Chemical Information and Modeling, 28, 31–36. Williams, J. A., Wassall, C. D., Kemple, M. D., & Wassall, S. R. (2013). An electron paramagnetic resonance method for measuring the affinity of a spin-labeled analog of cholesterol for phospholipids. The Journal of Membrane Biology, 246, 689–696. Yang, Z., Liu, Y., Borbat, P., Zweier, J. L., Freed, J. H., & Hubbell, W. L. (2012). Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution. Journal of the American Chemical Society, 134, 9950–9952.
Computer Modeling of Spin Labels: NASNOX, PRONOX, and ALLNOX Kathleen N. Beasley*,1, Brian T. Sutch*,1, Ma'mon M. Hatmal†, Ralf Langen{, Peter Z. Qin}, Ian S. Haworth*,{,2 *Department of Pharmacology & Pharmaceutical Sciences, University of Southern California, Los Angeles, California, USA † Department of Laboratory Medical Sciences, Faculty of Allied Health Sciences, Hashemite University, Zarqa, Jordan { Department of Biochemistry, University of Southern California, Los Angeles, California, USA } Department of Chemistry and Department of Biological Sciences, University of Southern California, Los Angeles, California, USA 1 These authors contributed equally to the work. 2 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. NASNOX 2.1 Protocol 3. PRONOX 3.1 Protocol 4. ALLNOX 4.1 Standard Label–Linker–Target Assemblies 4.2 Inclusion of a Custom Linker 4.3 Inclusion of a Custom Label and a Custom Target 4.4 Alternative Approaches for Complex Labels 4.5 Further Modifications of the Program References
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Abstract Measurement of distances between spin labels using electron paramagnetic resonance with the double electron–electron resonance (DEER) technique is an important method for evaluation of biomolecular structures. Computation of interlabel distances is of value for experimental planning, validation of known structures using DEER-measured distances, and determination of theoretical data for comparison with experiment. This requires steps of building labels at two defined sites on proteins, DNA or RNA; calculation of allowable label conformers based on rotation around torsional angles; computation of pairwise interlabel distances on a per conformer basis; and calculation of an average distance between the two label ensembles. We have described and validated two programs for this purpose: NASNOX, which permits computation of distances Methods in Enzymology, Volume 563 ISSN 0076-6879 http://dx.doi.org/10.1016/bs.mie.2015.07.021
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between R5 labels on DNA or RNA; and PRONOX, which similarly computes distances between R1 labels on proteins. However, these programs are limited to a specific single label and single target types. Therefore, we have developed a program, which we refer to as ALLNOX (Addition of Labels and Linkers), which permits addition of any label to any site on any target. The main principle in the program is to break the molecular system into a “label,” a “linker,” and a “target.” The user can upload a “label” with any chemistry, define a “linker” based on a SMILES-like string, and then define the “target” site. The flexibility of ALLNOX facilitates theoretical evaluation of labels prior to synthesis and accommodates evaluation of experimental data in biochemical complexes containing multiple molecular types.
1. INTRODUCTION Introduction of nitroxide radicals as spin labels at specific sites of biological macromolecules can provide information on molecular structure, flexibility, and environment using electron paramagnetic resonance (EPR). Several recent reviews have described techniques for site-directed spin labeling (Fielding, Concilio, Heaven, & Hollas, 2014; Hubbell, Lo´pez, Altenbach, & Yang, 2013; Jeschke, 2013) and subsequent analyses using spin-labeled proteins (Bordignon & Polyhach, 2013; Klare, 2013; Sahu, McCarrick, & Lorigan, 2013), DNA (Ding et al., 2014), RNA (Esquiaqui, Sherman, Ye, & Fanucci, 2014), and lipids (Gaffney, 2014; Schwarzmann, Arenz, & Sandhoff, 2014). Labeling of a target simultaneously at two sites permits determination of an interspin label distance using the double electron–electron resonance (DEER) method, as described in many of these reviews. The nitroxide radical of the spin label is commonly stabilized by its inclusion as a substituent of a multimethylated ring. The label is connected to the target molecule directly or via a flexible or rigid linker, with a wide variety of linker chemistries used to form the label–linker–target assembly. Some common examples are shown in Table 1, in which we use the “label– linker–target” nomenclature for a specific purpose, as described further below. The R1 and R5 assemblies (which include the same “label”: 1-oxyl-2,2,5,5-tetramethylpyrroline) are the most commonly used for proteins and DNA, respectively, with numerous examples in the literature (see above-referenced reviews). TOAC (a label containing a 1-oxyl-2,2,6,6tetramethylpiperidine ring) is increasingly commonly used as a rigid label in which the Cα of the protein backbone is also part of the label ring (Table 1). TOAC can be incorporated into short peptides for DEER measurements to probe peptide conformation (Milov, Tsvetkov, et al., 2014;
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Table 1 Examples of Spin Labels, Linkers, and Targets Linker Target Labela
a
Common Namesb
CSSC
Protein Cα
R1, MTSL, MTSSL
CS
DNA P
R5
CC
Thymine C5
5sp
None
Protein Cα
TOAC
None
Cytosine N4 Adenine N6
TEMPO
Cytosine N4 Adenine N6
TEMPO
Protein Cα
TOPP
L, linker; T, target. Short-hand names used in the literature to describe the label–linker–target assembly.
b
Sahu et al., 2014; Shabestari et al., 2014) and behavior at organic surfaces (Milov, Samoilova, et al., 2014). The piperidine ring in TOAC may also be used as a DNA label, as recently illustrated by Gophane and Sigurdsson (2015). In this context, the label is referred to as TEMPO and is connected directly or via a linker to a base target (amino group in cytosine or adenine) (Table 1). We return to this label–linker–target assembly in Section 4.3. Spin labels with a variety of
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structures have also been attached to molecules such as cyclodextrin (Krumkacheva et al., 2013), cholesterol (Williams, Wassall, Kemple, & Wassall, 2013), and stearic acid (Liu, Zhang, Fan, Wang, & Jiao, 2013; Reichenwallner & Hinderberger, 2013). The choice of labels may also depend on the experimental conditions, as discussed in a study of in-cell EPR using spin-labeled DNA quadruplexes (Holder, Drescher, & Hartig, 2013). In vivo considerations may become increasingly important, since EPR using spin-labeled stearic acid has been proposed as a diagnostic modality for colorectal cancer (Liu et al., 2013). In theoretical calculations of interspin label distances, accurate modeling of the label conformer distribution is necessary for labels connected to a target with a flexible linker. Three programs, MMM (Polyhach, Bordignon, & Jeschke, 2010), PRONOX (Hatmal et al., 2012), and MtsslWizard (Hagelueken, Ward, Naismith, & Schiemann, 2012) have been developed for computation of distributions of R1 on proteins (Table 1). These programs use distinct, but somewhat similar, approaches to calculation of rotamers in the R1 linker (Fig. 1A), based on information obtained from X-ray data (Guo, Cascio, Hideg, & Hubbell, 2008; Langen, Oh, Cascio, & Hubbell, 2000) and supported by quantum mechanical calculations (Warshaviak, Serbulea, Houk, & Hubbell, 2011). The programs give relatively similar results for interlabel distances based on a detailed evaluation by Jeschke (2013), with each having an RMSD of about 3 A˚ compared to experimental DEER distances. An accurate rotamer library is also available in the RosettaEPR package (Alexander et al., 2013). A direct comparison of theoretically generated label conformers with those obtained biophysically has also recently been described (Florin, Schiemann, & Hagelueken, 2014).
Figure 1 Definitions of rotatable torsional angles in the linkers of (A) the R1 protein label and (B) the R5 DNA label.
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In our development of PRONOX, we validated the program against a large database of experimental DEER distances obtained from R1-labeled proteins (Hatmal et al., 2012). We had previously developed a separate program, NASNOX, for similar use with R5-labeled DNA or RNA, and we have also tested NASNOX using experimental data (Price, Sutch, Cai, Qin, & Haworth, 2007). Both programs compute label distributions via torsional angle rotation in the linker joining the label to the protein or DNA backbone (Fig. 1), with subsequent calculation of all pairwise interconformer distances between the two label distributions and determination of an average interlabel distance. We typically quote this distance as that between the N atoms of the nitroxide groups (Hatmal et al., 2012; Price et al., 2007). The restriction of NASNOX and PRONOX to a single label type at a distinct biomolecular site is advantageous, in that it allows system-specific considerations to be implemented in the algorithms. Such considerations include label–biomolecule interactions and calculation of interlabel distances after weighting favorable conformers in the distribution, as described in Sections 2 and 3. However, the spin labeling field is expanding through use of multiple label types attached to various sites on proteins, DNA/ RNA, and other targets, using a variety of linkers through a range of chemistry. To facilitate calculations on these labels and to allow new labels to be examined theoretically prior to synthesis, we have developed a new program, entitled ALLNOX (Addition of Labels and Linkers). In this chapter, we first briefly describe use of NASNOX and PRONOX, but we mainly refer the reader to previous descriptions of these programs (Hatmal et al., 2012; Price et al., 2007; Qin et al., 2007). We then focus on utilization of ALLNOX for flexible building of spin-labeled biomolecules, automated calculation of label conformer distributions, and computation of interlabel distances. NASNOX, PRONOX, and ALLNOX are available at the URLs shown in Table 2.
Table 2 URLs for NASNOX, PRONOX, and ALLNOXa Program URL
NASNOX
http://pzqin.usc.edu/NASNOX/
PRONOX
https://ihlab.hsc.usc.edu/pronox/
ALLNOX
https://ihlab.hsc.usc.edu/allnox/
a
All the programs can be accessed from the “Programs” dropdown menu at https://ihlab.hsc.usc.edu/.
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2. NASNOX NASNOX is designed to model the conformational distribution of R5 at a DNA or RNA target site (Price et al., 2007; Qin et al., 2007). With the nucleic acid coordinates fixed, the program systematically varies torsion angles t1, t2, and t3 (Fig. 1B) and identifies allowed conformers that have no steric clash between the label and the target molecule. A clash is defined as a contact distance <75% of the sum of the van der Waals radii of the interacting atoms. Interspin label distances are calculated for every pairwise combination of labels in the allowed ensemble at the two sites, and the mean and standard deviation of the distances are then determined. Each R5 conformer distribution and the interspin label distances can be computed in less than a minute on a PC using the default settings. NASNOX uses the R5 label with a CS linker (Table 1) that connects to the DNA or RNA through formation of a phosphorothioate at the target site. An important feature of NASNOX is that a “site” can comprise two label distributions to allow for calculation of distances using spin labeling that result in a racemic mixture of labels at a given phosphodiester. A screenshot of the NASNOX site and a typical output structure are shown in Fig. 2. In this example, label conformer distributions (shown in green (light gray in the print version) and red (dark gray in the print version)) are calculated for labels with sulfur replacing each nonbridging oxygen atom of the phosphodiester at deoxynucleotide 18, and similarly at 36. The calculated interlabel distance is determined from the racemic mixture of labels at both sites. A single enantiomer distribution can also be calculated at a target site. We have described the use of NASNOX elsewhere (Qin et al., 2007) and the reader is directed to this description for further details. The following protocol is given in brief and with the goal of highlighting differences between NASNOX and ALLNOX.
2.1 Protocol Step 1 NASNOX is available as a Web application (http://pzqin.usc.edu/ NASNOX/). The initial screen does not contain default settings, so all numbers need to be entered by the user. Step 2 A target file in pdb format containing DNA or RNA must be uploaded using the “Choose File” button. Any other molecular types in the file will be ignored. The default in NASNOX is to add protons to the DNA or RNA if not already present. The
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Figure 2 Depiction of the NASNOX Web site and a structure resulting from a NASNOX calculation. The green (light gray in the print version) and red (dark gray in the print version) label distributions at each site (18 and 36) are for labels with sulfur replacing the respective nonbridging oxygen atoms at each phosphodiester.
two sites for labeling are entered in the corresponding boxes (Fig. 2: sites 18 and 36 in the example). If labeling at both nonbridging oxygen atoms (replaced by sulfur) is required, then each box “O1P” and “O2P” should be set to “1.” If a single enantiomer label distribution is required at, for example, O1P, then set the “O2P” box to “0.” Note 2.1 The identities of “O1P” and “O2P” in the pdb file are critical. The user should check this carefully, first to ensure that the atoms are named as such, and then to ensure that the correct enantiomer is being chosen. Note 2.2 NASNOX renumbers the input molecule; therefore, in this example, 18 and 36 are the 18th and 36th deoxynucleotides in the DNA. Step 3 The screenshot in Fig. 2 shows the default settings recommended in Price et al. (2007). Torsions t1 and t2 can adopt gauche(+), trans, and gauche() (60°, 180°, and 300°) positions (starting value 60°, three steps), and torsion t3 is varied from 0° to 330° in steps of 30° (starting value 30°, 12 steps).
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Note 3.1 The “Hydrophobic Contacts” option is unlikely to alter the results significantly, but this option might be useful if the label is likely to come into contact with a thymine methyl group. Note 3.2 The “Fine Search” option is unlikely to be needed for calculations on duplexes and other structures in which the label is not spatially limited. This option is useful in situations in which an initial run fails to locate an acceptable label conformer. The “Fine Search” option can be switched on for each torsion separately by setting the corresponding box to “1.” This option searches around the initial torsional setting (Price et al., 2007) to locate acceptable conformers. Using this option for all torsion angles will substantially increase the running time. Step 4 Click the “Submit” button. The results will appear on a new screen within a few seconds. A structure file (data001lig.pdb) and a text results file (data.add) can be downloaded. The structure file contains all the allowable label conformers, listed in order of site after the DNA or RNA coordinates. The results file includes details of the fate of each conformer (allowed or not allowed) and each pairwise distance (shown for N–N, O–O, and midpoint NO(mNO)–mNO distances) between allowed conformers at the two sites. The average N–N, O–O, and mNO–mNO distances and standard deviations are given at the end of the results file.
3. PRONOX PRONOX was developed for computation of interlabel distances between two R1-labeled protein sites (Table 1), based on calculation of spin label conformer distributions through variation of torsions χ1 to χ5 (Fig. 1A; Hatmal et al., 2012). Each label is added using the Cβ atom of the original amino acid (or guided by Hα for glycine). The geometry of the CSSC linker is based on crystallographic data (Langen et al., 2000). The nitroxide ring geometries are described in Hatmal et al. (2012), with reference to earlier work in Price et al. (2007). Conformers generated by rotation around χ1 to χ5 are accepted if they do not clash with the protein. A clash is defined as a label atom to protein atom distance of <75% of the sum of the van der Waals radii of the two atoms. This percentage is
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adjustable in the input and adjustment may be necessary for labels that are located in spatially restricted locations. An important aspect of PRONOX is the weighting of conformers that are known to be favorable based on X-ray evidence showing that the χ1 and χ2 torsions are well defined (Guo et al., 2008). The χ1, χ2 gauche(), gauche() (m,m); trans, gauche(+) (t,p); and trans, gauche() (t,m) conformers have a nonbonded interaction between Sδ and Hα ((m,m) and (t,p)) or other backbone atoms ((t,m)) and this makes these conformations favorable. Thus, the default in PRONOX is a mode in which conformers with these values for χ1 and χ2 are included in the distribution with a biased weighting (Hatmal et al., 2012). In the default setting, the torsional angle around the S–S bond (χ3) is taken to be 90° and all positions of χ4 and χ5 are considered to be isoenergetic. A screenshot of the PRONOX Web site and a typical output structure is shown in Fig. 3.
3.1 Protocol Step 1 PRONOX is available as a Web application (https://ihlab.hsc.usc. edu/pronox/). The initial screen is populated with all default settings. For most runs, all the user needs to enter are the two amino acid residue numbers (11 and 21 in the example in Fig. 3).
Figure 3 Depiction of the PRONOX Web application and a structure resulting from a PRONOX calculation. The R5 distributions are shown in orange (dark gray in the print version) and blue (dark gray in the print version) at sites 11 and 21, respectively.
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Step 2 A file in pdb format containing a protein should be uploaded. Any nonprotein atoms will be ignored. The default in PRONOX is not to add protons to the protein. Note 2.1 PRONOX renumbers the input protein; therefore, 11 and 21 are the 11th and 21st amino acids in the pdb input file. Step 3 The default settings for PRONOX should be satisfactory for labels that are not in a spatially restricted position. Torsions are defined using a starting value and a step number: for example, a starting value of 60° and a step of three gives torsional values of 60°, 180°, and 300°. Note 3.1 If the label is spatially restricted, the “vdW” setting can be modified to alter the clash criterion. We have found that a setting of 0.40 (the “low clash” mode) is effective for hindered labels (Hatmal et al., 2012). Note 3.2 The “Fine Search” option (as described for NASNOX) is also available for each torsion angle (by adding a check mark to the appropriate box) for labels in restricted locations. Note 3.3 For a spatially restricted label, the torsion increments can also be decreased, but we do not recommend this approach since it is not chemically justifiable. Step 4 We recommend that the default weighting is maintained in all runs. Note 4.1 Setting the “Weight” box to 0.5 will result in all labels being weighted equally. Step 5 If the target amino acid is a glycine, the “Glycine” box must be checked. All other boxes can be modified as in Steps 2–4. For addition of a label to glycine, the pdb file must contain a proton on the glycine Cα, and the proton must be named “HA3.” It is important to ensure that the correct prochiral proton is identified and named “HA3” in the pdb file. Step 6 Click the “Run PRONOX” button. The results will appear on a new screen within a few seconds. A structure file (output.pdb) and a text results file (data.add) can be downloaded. Note that these files will have a random computer-generated string in the file name after download. The structure file contains all the allowable label conformers, listed in order immediately after the amino acid of each labeled site in the pdb file. The results file includes details of each conformer (allowed or not allowed, favorable or unfavorable for
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weighting). After this listing for each site, the total number of favorable and unfavorable labels identified for the site is given, with a % in parentheses indicating how much each of these labels contributes to the conformer distribution. Each pairwise distance (N–N, O–O, and midpoint NO(mNO)–mNO) is shown between allowed conformers at the two sites. The average N–N, O–O, and mNO– mNO distances and standard deviations are given at the end of the results file. The total number of interlabel distances in the calculation is given in parentheses. Note 6.1 A useful feature of the PRONOX Web application is that each run has a designated job ID and results can be recovered using this ID. However, only a limited number of results are saved and there is currently no login enabled, so users should generally download results as they are obtained.
4. ALLNOX NASNOX and PRONOX are useful for computation of interlabel distances for R5-labeled DNA or RNA and R1-labeled proteins, respectively. However, these programs are limited in their restriction to a single label type and a single biomolecular target. To overcome this limitation, we have developed an algorithm that we refer to as ALLNOX. This program was written to allow the user to add a spin label with any chemistry to any site on any molecular target, with automated computation of label conformer distributions and calculation of interlabel distances based on these distributions. We begin by defining each labeled site as a “label–linker–target” assembly (Table 1). In this context, the “label” is the ring that contains the nitroxide radical (although this definition is not absolute); the “linker” is the chain of atoms between the label and the target, and is generally the source of conformational flexibility; and the “target” is the biomolecule (protein or DNA in the examples below, but this can be any molecule). In developing the ALLNOX Web application, we have tried to maintain the relative simplicity of the NASNOX and PRONOX Web applications and avoid the requirement for too many options. This is challenging given the need to accommodate user-defined label–linker–target assemblies that are unknown in advance.
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In the following sections, we describe use of ALLNOX for several different situations. In Section 4.1, we start with building of standard label– linker–target assemblies similar to those in NASNOX and PRONOX. In Sections 4.2 and 4.3, we illustrate the assembly design with increasingly more user-defined “unknown” elements.
4.1 Standard Label–Linker–Target Assemblies The standard label–linker–target assemblies that are currently implemented in ALLNOX are listed in Table 3, and are available from a dropdown menu on the ALLNOX Web site. We use common nomenclature to indicate these assemblies; for example, R1 and R5 are used to indicate the 1-oxyl-2,2,5,5-tetramethylpyrroline “label.” Some of the standard assemblies produce label–linker–target arrangements similar to those in NASNOX and PRONOX, but the calculated interlabel distances are not ˚ , which is within exactly the same; however, they are generally within 2 A the error of these calculations ( Jeschke, 2013). This is because in ALLNOX protons are not added to DNA (unlike in NASNOX) and no weighting of conformers is performed (unlike in PRONOX). 4.1.1 Protocol Step 1 ALLNOX is available as a Web application (https://ihlab.hsc.usc. edu/allnox/). The initial screen has input fields for the minimal options required for a run using standard label–linker–target assemblies at both sites. Table 3 Standard Label–Linker–Target Assemblies in ALLNOX Program Definition
R1-CSSC-aaCα
R1-labeled protein (similar to PRONOX)
R1-CSSC-GlyCα
R1-labeled protein at glycine (similar to PRONOX for glycine)
R5-CS-nucP
R5-labeled nucleotide (similar to NASNOX, connected via O1P)
R5-CC-ThyC5
Rigid label attached to thymine (5sp in Table 1)
TOAC(Cα)
TOAC with amino acid Cα in the label ring
TOPP-aaCα
TOPP-labeled protein (see Table 1)
TOPP-GlyCα
TOPP-labeled protein at glycine (see Table 1)
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Step 2 A pdb file containing the target (which can be any type of molecule) should be uploaded (Fig. 4A, red (dark gray in the print version) arrow). Most pdb files from common sources should work without alteration, other than elimination of unwanted chains. The default in ALLNOX is not to add protons. Currently, no protons should be included in the input pdb file. ALLNOX also renumbers input molecules (see Step 2 for NASNOX and PRONOX). Step 3 The next step is to define the label–linker–target assembly. Standard options (Table 3) are available via a dropdown menu for assemblies 1 and 2 (Fig. 4A, orange (light gray in the print version) arrows). In the example shown in Fig. 4A, the following assemblies were selected: “Label-linker-target 1”: “R1-CSSC-aaCα,” giving a PRONOX-like label at a protein site to be defined in Step 5. “Label-linker-target 2”: “R5-CS-nucP,” giving a NASNOXlike label at a nucleotide site to be defined in Step 5. Note 3.1 If the target amino acid is a glycine, “R1-CSSC-GlyCα” should be selected. Note 3.2 “R5-CS-nucP” assumes connection to DNA through replacement of the phosphodiester O1P atom with S. Attachment of labels (including R5) with other connectivity to DNA (including at the phosphodiester) is described in Section 4.3. Note 3.3 Other standard assemblies are discussed in Section 4.4. Step 4 The “vdW” boxes are defaulted to 0.75 as the recommended value. This value can be reduced if no results are produced in an initial run (see Step 3 for PRONOX). Step 5 The target sites are set by entering a residue number in each box (Fig. 4A, green (dark gray in the print version) arrows). This selection should reflect the choice of the label–linker–target assembly, as follows in the example in Fig. 4A: “Target Residue Number” 11 refers to the protein (or proteins) in the target pdb file because the standard label–linker–target (R1-CSSC-aaCα) includes a protein target. “Target Residue Number” 18 refers to the DNA in the target pdb file because the standard label–linker–target (R5-CS-nucP) includes a DNA target.
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Figure 4 Depiction of the input fields on the ALLNOX Web application and a structure output for an ALLNOX run using standard label–linker–target assemblies at both sites. (A) Selections for the target, label–linker–target assemblies, target residue numbers, and conformation are indicated by red (dark gray in the print version), orange (light gray in the print version), green (dark gray in the print version), and blue (dark gray in the print version) arrows, respectively. (B). Table of default linker torsions that can be edited by the user. This table is generated in “Manual” conformation mode. Each torsional variation is defined by a minimum and maximum value and an increment. The four atoms comprising the torsional angle are shown on the right. Atom names in the linker are indicated by “L” as the last character. (C). A structure (output.pdb) resulting from an ALLNOX calculation for a protein–DNA complex, showing the R1 label distribution on amino acid 11 (yellow (light gray in the print version)) and the R5 label distribution on deoxynucleotide 18 (blue (dark gray in the print version)).
Note 5.1 ALLNOX renumbers the input molecule; therefore, in this example, 11 is the 11th amino acid in the protein and 18 is the 18th deoxynucleotide in the DNA. Step 6 The last selection before running the simulation is to choose a conformation mode (Fig. 4A, blue (dark gray in the print version) arrow). The following options are available:
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Static: Static mode will generate a single fixed conformation for each label–linker assembly and will ignore clashes with the target. This mode is useful as an initial check, particularly for a user-defined label–linker–target assembly (see Sections 4.2 and 4.3). Automatic: This is the default mode, in which ALLNOX generates a conformational distribution of the label–linker system. Rotatable torsions (single bonds) are varied as gauche(+) (60°), trans (180°), and gauche() (300°), except for the torsion around an S–S bond (90°, as in PRONOX). Manual: This mode will generate a table of the default linker torsions that can be freely edited by the user (Fig. 4B). Atoms in each label–linker–target assembly are shown at the top of this page. Each torsional variation is defined by a minimum and maximum value and an increment. The four atoms comprising the torsional angle are also shown on the right. Atom names in the linker are written as the character in the input linker string, the position of the atom in the string (1 for the atom closest to the label), and “L” as the last character. Note 6.1 In “Static” or “Automatic” mode, clicking the “Run” button will initiate the ALLNOX calculation of the label–linker–target assemblies and interlabel distances. Note 6.2 In “Manual” mode, clicking the “Run” button will produce the editable table of torsion angles. Once this is edited (or simply checked for correctness), clicking the “Run” button again will initiate the ALLNOX calculation. Note 6.3 In “Manual” mode, a torsion can be fixed by entering the same minimum and maximum value and including any positive number (do not enter 0) as the increment. Step 7 The results will appear on a new screen within a minute. A structure file (“output.pdb,” depicted in Fig. 4C) and a text results file (data. add) will be generated and can be downloaded. These files will have a random computer-generated string in the file name after download. The structure file contains all allowable label conformers, listed after the contents of the target pdb file. The results file includes details for each conformer, the total number of conformations found for each label–linker assembly, the pairwise N–N and O–O (nitroxide group) distances between all allowed conformers at the two sites, and the average N–N and O–O distances at the
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end of the file. The total number of interlabel distances in the calculation is given in parentheses. Note 7.1 Each run has a designated job ID and results can be recovered using this ID. However, only a limited number of results are saved and there is currently no login enabled, so users should generally download results as they are obtained. Note 7.2 User registration and account creation will be available in a forthcoming version of ALLNOX, allowing users to perform advanced calculations and save run data on the server.
4.2 Inclusion of a Custom Linker There are a relatively small number of labels used in spin labeling (although this number is growing) and differences in labeling approaches often lie in linker variation. To accommodate modeling of standard labels linked to standard targets through a “non-standard” (custom) linker, ALLNOX permits the user to enter the linker chemistry in a format that is similar to a limited and slightly altered SMILES (simplified molecular-input line-entry system) format (Weininger, 1988). The current limits on the linker are a maximum of 10 atoms with no branches, including carbon (C or c), oxygen (O), nitrogen (N or n), sulfur (S), and phosphorus (P): upper case C or N indicates an sp3 C atom or an amine N, and lower case c or n indicates sp2 C or amide N. Functional groups and bonds (for which the geometry should be correct) based on these combinations are ether, thioether, amine, amide, alkene, and S–S, O–P, and S–P bonds. The following protocol should be read with additional reference to Section 4.1. 4.2.1 Protocol Step 1 To initiate customization of a label–linker–target assembly, the user chooses “Custom” in the dropdown menu below “Label-LinkerTarget 1” or “Label-Linker-Target 2” (Fig. 5A, red (dark gray in the print version) arrows). This selection generates two new dropdown menus under titles of “Label Selection” (Fig. 5A, orange (light gray in the print version) arrows) and “Target Selection” (Fig. 5A, green (dark gray in the print version) arrows), and a “Linker” box (Fig. 5A, blue (dark gray in the print version) arrow). Step 2 The user may select a standard label or a custom label (see Section 4.3). In the example in Fig. 5A, a R1(R5) standard label
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Figure 5 Depiction of the input fields for the ALLNOX Web application and a structure output for an ALLNOX run using standard labels and targets, and a custom linker at both sites. (A) Selections for the label–linker–target assemblies, labels, targets, and linker boxes are identified by red (dark gray in the print version), orange (light gray in the print version), green (dark gray in the print version), and blue (dark gray in the print version) arrows, respectively. (B) Table of default linker torsions. (C) A structure (output.pdb) resulting from an ALLNOX calculation for a protein–DNA complex, showing the label distributions with custom linker CCSSC on amino acid 11 (green (dark gray in the print version)) and custom linker CncCS on deoxynucleotide 18 (pink (dark gray in the print version)).
is chosen at both sites (Fig. 5, orange (light gray in the print version) arrows). Note 2.1 R1(R5) indicates the 1-oxyl-2,2,5,5-tetramethylpyrroline ring (Table 1). We use the R1(R5) terminology because R1 is typically used to describe this label when connected (via a CSSC linker) to proteins, whereas R5 is used when the same ring is connected (via a CS linker) to DNA or RNA. Step 3 The user may also select a standard target or a custom target (see Section 4.3). In the example in Fig. 5A, an aaCα standard target
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(Cα for any amino acid except glycine) is chosen for label–linker– target assembly 1, and a nucP standard target (nucleotide phosphodiester P) is chosen for label–linker–target assembly 2 (Fig. 5A, green (dark gray in the print version) arrows). Step 4 The custom linker is entered in the “Linker” box for each label– linker–target assembly (Fig. 5A, blue (dark gray in the print version) arrow). In entering the linker sequence, it is important to understand that the left most atom will be attached to the label and the right most atom will be attached to the target. In the example in Fig. 5A, a CCSSC linker is used for label–linker–target assembly 1 (that is, an additional C atom compared to the standard linker in the R1-CSSC-Cα assembly) and a CncCS linker is used for label–linker–target assembly 2 (i.e., inclusion of an additional C and an amide compared to the standard linker in the R5-CS-nucP assembly). Note 4.1 ALLNOX can accommodate linker sequences up to a maximum of 10 heavy atoms with single and double bonds, but currently excluding branches. See above for allowable linkers. Note 4.2 A more complete SMILES format (Weininger, 1988) for entry of the linker atoms will be available in a forthcoming version of ALLNOX. Note 4.3 When running a custom linker sequence with >five atoms, the run may take several minutes to complete. Step 5 The “Manual” mode is recommended if a custom linker is used. This allows the user to check that the torsional angles have been set correctly in the editable table. This table is shown in Fig. 5B for the example used in this protocol. ALLNOX recognizes the amide bond and does not include this as a rotatable torsion. Step 6 The results are presented as described in Step 7 of Section 4.1. The output file for the example described in this section is depicted in Fig. 5C.
4.3 Inclusion of a Custom Label and a Custom Target The ultimate goal of ALLNOX is to allow the user to build a custom “label” connected via a user-defined “linker” to a custom “target” (Fig. 6). This requires the user to upload a structure file for the label and to define the topologies and geometries of the label–linker and linker–target connections.
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This approach is facilitated by additional options on the ALLNOX Web site, as described below. We have tried to minimize the amount of information requested of the user and the current program is capable of understanding the chemistry of many label–linker and linker–target connections. This will be improved as we obtain feedback on the functionality of the program. In this section, we use the example of the TEMPO label–linker–target assembly with an amide linker and a cytosine N4 (amino group) target (Table 1; Fig. 7B). Synthesis of this assembly was recently described by Gophane and Sigurdsson (2015). To include this label in ALLNOX, a pdb file for the TEMPO ring (Table 1) was generated from the following SMILES string: C1C(C)(C)N(¼O)C(C)(C)CC1. This example illustrates how ALLNOX can be used to generate a model of a custom label– linker–target assembly. Understanding of the utilization of the program
Figure 6 Depiction of the input fields for an ALLNOX run using a custom assembly. Selections for the label–linker–target assemblies, labels, targets, label atom numbers, target atom names, label to linker geometries, and linker to target geometries are indicated by red (dark gray in the print version), orange (light gray in the print version), black, green (light gray in the print version), yellow (light gray in the print version), blue (dark gray in the print version), and pink (dark gray in the print version) arrows, respectively. The structure shows the atom names used to define the geometry between the label (red (dark gray in the print version)) (k, j, and i) and linker (green (light gray in the print version)) (h) and between the linker (green (light gray in the print version)) (x, y, and z) and target (blue (dark gray in the print version)) (a, b, and c). Note that atoms “i,” “j,” “k” and “a,” “b,” “c” are shown as bonded atoms, but this is not a requirement.
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Figure 7 (A) Edited torsion angle table for the TEMPO-amide-cytosine N4 label–linker– target assembly. Torsions t1 and t2 are set at 180° for both assemblies (sites 19 and 6). (B) Structure of the label–linker–target assembly showing the positions of torsions t1, t2, and t3. (C) Result of an ALLNOX calculation at site 19, showing three conformers (red (dark gray in the print version), green (light gray in the print version),and blue (dark gray in the print version)) generated for the three positions (60°, 180°, and 300°) for torsion t3.
for the complete user-defined assembly may require initial reading of Sections 4.1 and 4.2. 4.3.1 Protocol Step 1 To initiate custom upload of a pdb file containing a custom label, the user first chooses “Custom” in the dropdown menu below “Label-Linker-Target 1” or “Label-Linker-Target 2” (Fig. 6, red (dark gray in the print version) arrows), and then chooses “Custom” again in the “Label Selection” dropdown menu (Fig. 6, orange (light gray in the print version) arrows). Step 2 The selection in Step 1 reveals a “Choose File” upload button, with which the pdb file for the label can be uploaded (TEMPU_UC.pdb in the example in Fig. 6). Note 2.1 The pdb file for the “label” may be generated from a SMILES string using a Web site such as “SMILES
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Translator” (http://cactus.nci.nih.gov/translate/; settings: “PDB,” “3D,” upload the SMILES string as a .txt file). Note 2.2 The PubChem Web site (https://pubchem.ncbi.nlm. nih.gov/) or commercial sites such as that at Santa Cruz Biotechnology (http://www.scbt.com/chemicalstable-spin_traps_spin_probes_and_spin_labels.html) are useful sources of SMILES strings for spin labels. Note 2.3 Make sure that the nitroxide group in the SMILES string is represented as N¼O. This ensures the correct geometry in the resulting pdb file (with use of NO, the oxygen is interpreted as a hydroxyl group). Note 2.4 Some editing of the pdb file (deletion of atoms, such as protons at the atom connecting to the linker) may be needed to produce the appropriate file for input into ALLNOX. Note 2.5 ALLNOX will recognize the nitroxide group automatically. No specific atom names are required in the pdb file. Step 3 The “Linker” boxes should be completed (see Step 4 in Section 4.2). Step 4 The selection in Step 1 produces “Label Atom Number” boxes (Fig. 6, green (light gray in the print version) arrow) in which atoms “i,” “j,” and “k” in the label can be defined. An illustration depicting the location of these atoms is shown in Fig. 6. The numbers for “i,” “j,” and “k” should correspond to the order of atoms in the label pdb file. Note 4.1 Atoms “i,” “j,” and “k” are any three atoms in the label, with “i” connected to the linker (or to the target, if there is no linker). Atom “h” is the first atom of the linker. Step 5 The selection in Step 1 reveals “Label to Linker Geometry” boxes (Fig. 6, blue (dark gray in the print version) arrow). These allow definition of the bond length (distance) “i–h,” angle “j–i–h,” and torsion “k–j–i–h” (see structure in Fig. 6 for definition of atoms “k,” “j,” “i,” and “h”). ALLNOX will make a best guess at these geometries. The values can be edited by the user. Step 6 In the “Target Selection” boxes (Fig. 6, black arrows), the user can choose a standard target or a custom target. If a standard target is chosen, no further input is required. If a custom target is chosen,
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two new boxes appear: “Target Atom Name” (Fig. 6, yellow (light gray in the print version) arrow) and “Linker to Target Geometry” (Fig. 6, pink (dark gray in the print version) arrow). Step 7 In the “Target Atom Name” boxes (Fig. 6, yellow (light gray in the print version) arrow) the atom names for atoms “a,” “b,” and “c” in the target can be defined. An illustration depicting the location of these atoms is shown in Fig. 6. An atom name should be entered in the “Replacement” box to indicate if an atom within the target is going to be replaced by an atom in the linker (or in the label if there is no linker). This box can be left blank if no target atom is to be replaced (as in the example of the TEMPO-derived spin label). Step 8 The “Linker to Target Geometry” boxes (Fig. 6, pink (dark gray in the print version) arrow) allow definition of the bond length (distance) “z–a,” angle “z–a–b,” torsion “z–a–b–c,” angle “y–z–a,” torsion “y–z–a–b,” and torsion “x–y–z–a” (see structure in Fig. 6 for definition of atoms “x,” “y,” “z,” “a,” “b,” and “c”). ALLNOX will make a best guess at these geometries. The values can be edited by the user. Note 8.1 Atoms “a,” “b,” and “c” are any three atoms in the target, with “a” connected to the linker (or to the label, if there is no linker). Atoms “x,” “y,” and “z” are the last three atoms of the linker (“z” is the last atom). Step 9 The “Manual” conformation mode should be used to check that the correct linker torsion angle variations have been defined and to make changes (see Step 5 of Section 4.2). Step 10 The results are presented as described in Step 7 of Section 4.1. The results of the protocol for the TEMPO-amide-cytosine N4 label– linker–target assembly are shown in Fig. 7. First, we note that this is an example in which the program does not have information on the linker to target geometry of the urea group formed in the assembly (Fig. 7B). Thus, the original torsion table had minimum, maximum, and increment values of 60°, 300°, and 120°, respectively. The torsion table in Fig. 7A is shown after editing of torsions t1 and t2 (for both label–linker–target assemblies) such that these torsions are fixed at 180°. This is an example of an assembly that requires some user intervention, but this is straightforward using the Web site. With this change, ALLNOX produces label conformers at both assembly sites. Three conformers are found at site 19 (Fig. 7C).
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4.4 Alternative Approaches for Complex Labels Some spin labels do not fit into the label–linker–target paradigm used in the protocols described above. However, such labels can be accommodated in ALLNOX. The simplest approach is to include the “linker” as part of the uploaded pdb file for the “label.” For a label such as sp5 (Table 1) this is acceptable since the “linker” is a triple bond that is clearly rigid. For TOPP (Table 1), the 1-oxyl-2,2,6,6-tetramethyl-3,5-dioxopiperazine “label” is connected through an aromatic ring to a Cα carbon. This label can be included as part of a standard label–linker–target assembly in the dropdown menu (Table 3), but rotation of the nitroxide-containing ring with respect to the aromatic ring cannot currently be performed. Finally, we note that inclusion of a racemic mixture of R5 (or other labels) at a DNA phosphodiester (which is possible for R5 in NASNOX) is not implemented as a standard label–linker–target assembly in the version of ALLNOX described here. Incorporation of this option is among the planned modifications of the program.
4.5 Further Modifications of the Program We plan further developments of ALLNOX that include increasing the degree of automation of calculation of label conformer distributions; increasing flexibility in linker definition (e.g., including branched linkers); weighting of conformer distributions based on calculation of label–target interactions, target flexibility, and expanding the program to include other targets. One particular area of interest is the growing number of bifunctional spin labels, as illustrated by bisMTSL ( Jeschke, 2013) and the novel DNAtargeted R5c label (Nguyen, Popova, Hideg, & Qin, 2015), both of which have two linker arms. There is potential for use of ALLNOX in experimental design using these labels, based on computation of label conformers through variation of one arm of the label combined with a search algorithm to find possible reactive positions for the second arm. The methods described in this chapter for nitroxide spin labels are also applicable to other spin labels, such as chelated Gd3+ ions (Goldfarb, 2014) and trityl labels (Reginsson, Kunjir, Sigurdsson, & Schiemann, 2012; Shevelev et al., 2014; Yang et al., 2012).
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