- Email: [email protected]
The Preparation of 19F-Labeled Proteins for NMR Studies
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natural BPTI, when water soluble, form conformational ensembles that favor nativelike structure. To produce a native state mimic, we incorporate two or more core modules into larger peptides in which module–module interactions are promoted. The interactions are expected to be mutually stabilizing, and thus to further shift the conformational bias of component core modules toward more ordered structure. This idea has been realized in the protein BetaCore, in which two core modules are incorporated into a single molecule by means of a long cross-link.36,37 BetaCore is monomeric in water and forms a new fold composed of a four-stranded, antiparallel -sheet. The single, dominant conformation of BetaCore has been characterized by various NMR experiments. Acknowledgments We thank Andrew Robertson and D. Wayne Bolen for critical reading of the manuscript and helpful discussions. This work is currently supported by NIH Grant GM51628. 36 37
N. Carulla, C. Woodward, and G. Barany, Protein Sci. 11, 1539 (2002). N. Carulla, G. Barany, and C. Woodward, Biophys. Chem. 101–102, 67 (2002).
[18] The Preparation of 19F-Labeled Proteins for NMR Studies By Carl Frieden, Sydney D. Hoeltzli, and James G. Bann Introduction
The incorporation of 19F-labeled amino acids into proteins for nuclear magnetic resonance (NMR) spectroscopy has been a technique used for many years as a probe of protein structure and dynamics. Three previous articles in Methods of Enzymology1–3 have dealt with this subject. Two recent excellent reviews4,5 describe the advantages of using 19F-NMR for such studies and cover the field well, pointing out the usefulness of fluorine as a probe of local environment. The present chapter discusses methods of incorporating 19F-labeled amino acids into proteins. 1
B. D. Sykes and W. E. Hull, Methods Enzymol. 49, 271 (1978). G. Horton and I. Boime, Methods Enzymol. 96, 777 (1983). 3 J. T. Gerig, Methods Enzymol. 177, 3 (1989). 4 M. A. Danielson and J. J. Falke, Annu. Rev. Biophys. Biomol. Struct. 25, 163 (1996). 5 J. T. Gerig, in ‘‘Biophysical Textbook Online’’ (2001). www.biophys.org/btol/ 2
METHODS IN ENZYMOLOGY, VOL. 380
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As noted in the two recent reviews,4,5 there are many properties of fluorine that make it a useful probe for studies of protein structure and function. For example, the fluorine nucleus is small, only slightly larger than the hydrogen nucleus. Although the dipole moment is considerably different, it is expected that there will be a minimal perturbation to the structure, stability, and functionality of the protein. Fluorine does not occur naturally in proteins so that a protein labeled with an 19F-labeled amino acid will exhibit NMR peaks due only to the label. Because fluorine is extraordinarily sensitive to its environment, and to local shielding effects,6 the NMR peaks are typically well resolved from one another in a one-dimensional (1D) NMR spectrum. This sensitivity makes 19 F-NMR extremely well suited for studies of both protein structure and protein folding, since even in the denatured state the peaks are frequently resolved.7–10 It should also be noted that 19F-NMR can be used to examine structural aspects of much higher molecular weight protein than are generally accessible by proton NMR. Under conditions in which two or more conformers exist in equilibrium, it may be possible to use 19F-NMR to calculate rate or dissociation constants if the conformers have different chemical shifts. If, for example, two different conformers exchange slowly on the NMR time scale, there will be two peaks and dissociation constants can be determined by quantifying the area of each peak. For conformers or liganded forms in intermediate exchange on the NMR time scale (roughly 5–5000 s1 depending upon chemical shift difference), the exchange rate kex may be determined by lineshape analysis.11 For conformers or liganded forms in slow exchange, in the millisecond range, the exchange rate may be accessible through two-dimensional experiments such as nuclear Overhauser enhancement spectroscopy (NOESY). Finally, the accessibility of a residue to bound or bulk water can be assessed by 19F-1H HOESY experiments.12 When using 19F-NMR to study protein folding, two major types of experiments can be carried out: an equilibrium measurement that involves the determination of peak chemical shift and intensity as a function of denaturant concentration or a kinetic experiment measuring peak intensity as a function of time after dilution of the denaturant. This latter can be done by manual mixing or, for faster reactions (1 s or longer), using a 6
E. Y. Lau and J. T. Gerig, Biophys. J. 73, 1579 (1997). S. D. Hoeltzli and C. Frieden, Biochemistry 33, 5502 (1994). 8 S. D. Hoeltzli and C. Frieden, Biochemistry 35, 16843 (1996). 9 S. D. Hoeltzli and C. Frieden, Biochemistry 37, 387 (1998). 10 J. G. Bann, J. Pinkner, S. J. Hultgren, and C. Frieden, Proc. Natl. Acad. Sci. USA 99, 709 (2002). 11 J. Sandstrom, ‘‘Dynamic NMR Spectroscopy.’’ Academic Press, New York, 1982. 12 D. P. Cistola and K. B. Hall, J. Biomol. NMR 5, 415 (1995). 7
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Fig. 1. 19F-NMR spectra of apo, binary, and ternary complexes of E. coli dihydrofolate reductase labeled with 6-19F-tryptophan. The data were collected at 22 on a Varian VXR 500 equipped with a Nalorac indirect detection probe. The five resonances were assigned using site-directed mutagenesis. MTX is the dihydrofolate analog, methotrexate. Data taken from Hoeltzli and Frieden.7
stopped-flow device.13 Because unfolded chemical shifts are frequently resolved, it is also possible to assess each residue individually in the unfolded state. Note that these measurements are different from those of hydrogen/ deuterium exchange in that the latter measures the ability of backbone amide protons to exchange with the solvent while the 19F-NMR experiments assess the environment of a specific amino acid side chain. Figure 1 illustrates a typical equilibrium experiment with native Escherichia coli dihydrofolate reductase. In this figure, the five tryptophan residues have been substituted with 6-19F-tryptophan. There are five clearly separated peaks for the native protein. Addition of ligands such as NADP or methotrexate (MTX) affects some peaks more dramatically than others. Four peaks are observed in the denatured protein (data not shown). In the presence of urea, all peaks in the native protein, except for Trp-22, are in slow exchange with the denatured form. In a technological advance, a fluorine cryoprobe (Varian) is currently available allowing either fewer transients or lower protein concentrations to be used for a given signal-to-noise (S/N) ratio. In our experience the S/N ratio is at least 4-to 5-fold greater than the conventional probes currently in use. This is particularly important for studies of proteins that tend 13
S. D. Hoeltzli, I. J. Ropson, and C. Frieden, in ‘‘Techniques in Protein Chemistry,’’ pp. 455–465. Academic Press, New York, 1994.
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Fig. 2. 19F-NMR spectra of 6-19F-tryptophan–labeled PapD recorded as a function of time after a stopped-flow urea jump from 4.5 M urea to 2.25 M urea. The final concentration of protein was 70 M using a fluorine cryoprobe on a Varian Unity-Plus 500-MHz spectrometer with 32 transients at each time point. Data were obtained at 20 as described by Bann et al.10 The buffer was 30 mM Mops/HCl, pH 7.0.
to aggregate at higher concentrations, or for detecting peaks that may have previously been difficult to quantify. Figure 2 shows a kinetic experiment using 70 M of the protein PapD and the fluorine cryoprobe. PapD, a bacterial chaperone, is a two-domain protein in which each domain has one tryptophan. In these experiments we used stopped-flow methods as described elsewhere.7–10 As shown by Fig. 2, changes in peak intensity can be measured as a function of time after dilution of the denaturant. Interestingly, an intermediate form, represented by the peak at 45.5 ppm, is present during refolding and disappears as the protein finishes the folding process. Although not shown in Fig. 2, it should be pointed out that the earliest time one can collect a spectrum after diluting out the denaturant is about 1–2 s. Unfortunately, there is a least one problem that has not been solved: it has not been easy, so far, to relate the chemical shift of the fluorine signal to the structural environment surrounding the fluorine within the protein.5,14 Therefore, it is not yet possible to predict the chemical shifts in a 19 F-NMR spectrum based on analysis of the protein’s amino acid sequence or the analysis of its three-dimensional structure. Consequently, to assign 14
J. G. Pearson et al., Biochemistry 36, 3590 (1997).
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the peaks from the labeled amino acids in a spectrum, one needs to use sitedirected mutagenesis. Techniques used for this method will be outlined in detail in this chapter. Currently, there are a number of different analogs that can be used to label proteins biosynthetically, and the list of available amino acids continues to grow as improved methods for the efficient syntheses of these amino acids continue.15 Most studies thus far have utilized aromatic amino acids for reasons discussed later. Incorporation of
19
F-Labeled Amino Acids into Proteins
For production of proteins containing 19F-labeled amino acids, biosynthetic incorporation of the labeled amino acids by microbial protein expression is the strategy of choice. High yields of >90% labeled protein are possible and biosynthetic methods are cost effective relative to chemical or in vitro synthesis. High levels of incorporation are important since this allows lower protein concentrations to be used and avoids a heterogeneous population of labeled protein. A number of approaches have been successfully utilized to produce very high label incorporation and two common themes emerge: (1) the importance of placing the gene for the protein of interest under control of a tightly regulated inducible promoter and (2) the critical issue of completely depleting and preventing synthesis of, natural amino acids from the labeling media prior to inducing protein production. Because 19F-labeled amino acids can inhibit bacterial growth to differing degrees16 the bacteria usually cannot be grown from inoculation on fluorine-containing medium. Bacterial cultures can be grown on defined medium containing a limited amount of the unlabeled amino acid and then either grown to the point where unlabeled amino acid is completely depleted prior to introduction of the 19F-labeled amino acid (for example, Kranz et al.17) or harvested and transferred to defined medium containing 19 F-labeled amino acid. Our laboratory has obtained more consistent results for a number of different proteins with the latter approach. It should be noted that some proteins cannot be produced under conditions that allow complete labeling (for example, Luck and Falke18), and in these cases it may be necessary to accept a lower level of fluorine label incorporation by adding unlabeled amino acid to the labeling medium or by incompletely depleting the unlabeled amino acid. 15
A. Sutherland and C. L. Willis, Nat. Prod. Rep. 17, 621 (2000). R. E. Marquis, in ‘‘Handbook of Experimental Pharmacology’’ (F. A. Smith, ed.), Vol. 20. pp. 165–192. Springer-Verlag, Berlin, 1970. 17 J. K. Kranz, J. Lu, and K. B. Hall, Protein Sci. 5, 1567 (1996). 18 L. A. Luck and J. J. Falke, Biochemistry 30, 4257 (1991). 16
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Labeling with Aromatic Amino Acids To date, most studies of 19F-labeled proteins have involved incorporation of 19F-labeled aromatic amino acids. There are several reasons for this. Usually, even quite large proteins contain a limited number of aromatic amino acids, simplifying the problem of assignment. Several healthy strains of bacteria auxotrophic for each of the aromatic amino acids are readily available. In addition, m-19F-tyrosine, 4-, 5-, and 6-19F–labeled tryptophan, and m-, o-, and p-19F-phenylalanine are all commercially available at reasonable cost. All of these analogs have been used sucessfully in studies of various proteins. In some studies, more than one analog has been successfully incorporated into the same protein.19–22 These results and those from our laboratory (Ropson and Frieden, unpublished observations; Hoeltzli and Frieden, unpublished observations) suggest that proteins labeled with different analogs can express at different levels, can differ in chemical shift resolution, and might differ in structural perturbation and stability. Examination of the crystal structure and the local contacts that are likely to be affected by the substitution will certainly help guide the experimentalist in the choice of an analog. It may be necessary to first try a series of analogs to determine if there is an effect on stability. In the absence of a comprehensive theory predicting fluorine chemical shifts the choice of analog remains largely empirical. Use of Nonauxotrophic Strains. The simplest approach to incorporation of a 19F-labeled amino acid is to use whatever nonauxotrophic bacterial strain has been found to give good protein expression and to repress endogenous amino acid synthesis with high concentrations of amino acids. This approach is limited by the fact that 19F-labeled amino acids are generally inhibitory to bacterial growth. However, Lu and co-workers23 achieved incorporation of greater than 90% 3-19F-tyrosine into lac repressor protein by using high concentrations of tryptophan, phenylalanine, and tyrosine to repress 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase, the first enzyme of the aromatic pathway. Strain CSH46 (also known as 96) was grown in M9 minimal media supplemented with 1% glucose, 1 g/ml thiamine, and 0.2 mM amino acids except tyrosine, tryptophan, and phenylalanine. Tryptophan and phenylalanine were supplemented at 1 mM, tyrosine was omitted, and the cells grown to A550 nm of 1.0. The cultures 19
G. S. Rule, E. A. Pratt, V. Simplaceanu, and C. Ho, Biochemistry 26, 549 (1987). C. Lian et al., Biochemistry 33, 5238 (1994). 21 M. A. Dominguez, Jr., K. C. Thornton, M. G. Melendez, and C. M. Dupureur, Proteins 45, 55 (2001). 22 C. Minks, R. Huber, L. Moroder, and N. Budisa, Biochemistry 38, 10649 (1999). 23 P. Lu, M. Jarema, K. Mosser, and W. E. Daniel, Proc. Natl. Acad. Sci. USA 73, 3471 (1976). 20
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were shifted to 42 to induce production of lac repressor and 1 mM 3-19F-tyrosine added. Use of a nonauxotrophic strain to incorporate 19 F-labeled amino acids has resulted in lower degrees of label incorporation in other reports.21,24 Careful attention to optimizing the inhibition of amino acid synthesis and the concentration of labeled amino acid is probably critical to success. Whether or not high (>90%) levels of label incorporation can be obtained, this approach may be valuable when expression of the protein of interest requires specific properties not available in an existing auxotrophic strain. A second approach is the use of glyphosate to induce aromatic amino acid auxotrophy in a nonauxotrophic bacterial strain. Glyphosate is an inhibitor of 5-enolpyruvylshikimic acid 3-phosphate biosynthesis and suppresses the production of aromatic metabolites, including the aromatic amino acids tryptophan, tyrosine, and phenylalanine.25 This strategy may be especially useful when an amino acid auxotroph of desirable properties is not available, or when the protein of interest cannot be expressed in available auxotrophs.26 Recently, complete 5-fluorotryptophan labeling of T. maritima cold shock protein using glyphosate to repress aromatic amino acid synthesis has been reported by Schuler et al.27 Briefly, cells were grown at 37 in minimal medium containing 50 mg/liter of all amino acids except tryptophan. Aromatic amino acid synthesis was suppressed with 1 g/liter glyphosate. When the cultures left exponential growth at A550 nm 1.5, 50 mg/liter dl-5-19F-tryptophan was added and protein expression induced for 4 h prior to harvest. Use of glyphosate has resulted in lower levels of incorporation in other reports.28 Depletion of the unlabeled amino acid from the growth medium is critical and needs to be monitored carefully. Use of Auxotrophic Strains. Most studies of 19F-labeled amino acids incorporated into proteins have utilized bacterial strains auxotrophic for the amino acid of interest. Numerous aromatic amino acid auxotrophs are available from individual investigators, through the ATCC or the Yale-New Haven E. coli Genetic Stock Center. The properties and sources of several auxotrophs we have found to be particularly useful are listed in Table I. Generally, the auxotrophic bacteria are transformed with a plasmid encoding the gene for the protein of interest under the control of a tightly regulated promoter using standard techniques. Transformed bacteria are grown on rich or defined medium containing a specific concentration of 24
C. M. Dupureur and L. M. Hallman, Eur. J. Biochem. 261, 261 (1999). L. Comai, L. C. Sen, and D. M. Stalker, Science 221, 370 (1983). 26 P. Bai, L. Luo, and Z. Peng, Biochemistry 39, 372 (2000). 27 B. Schuler, W. Kremer, H. R. Kalbitzer, and R. Jaenicke, Biochemistry 41, 11670 (2002). 28 H. W. Kim, J. A. Perez, S. J. Ferguson, and I. D. Campbell, FEBS Lett. 272, 34 (1990). 25
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TABLE I Useful E. COLI Auxotrophsa Strain
Auxotrophy
Mutation
Reference
W3110TrpA33 DL39 (CGSC #6913) NK6024 (CGSC #6178)
Trp Asp, Ile, Leu, Phe, Tyr Phe
trpA33 tyrB pheA::Tn10 (tetR)
40 30 31
a
Strains are available from the E. coli genetic stock center (http://cgsc.biology.yale.edu).
the natural amino acid of interest, then harvested and resuspended in defined medium containing the fluorine-labeled amino acid. The cells are allowed to recover for a period of time sufficient to deplete intracellular stores of unlabeled amino acid and protein production is then induced for an optimal period of time and cells harvested. Using this method, we have successfully produced 6-19F-tryptophan–labeled rat intestinal fatty acid binding protein,29 6-19F-tryptophan8,9 and p-19F-phenylalanine–labeled E. coli dihydrofolate reductase (Hoeltzli and Frieden, unpublished observation), 6-19F-tryptophan10 and p-19F-phenylalanine–labeled E. coli PapD (Bann and Frieden, unpublished data), and 6-19F-tryptophan–labeled murine adenosine deaminase (Shu and Frieden, unpublished data), with greater than 90% incorporation of label as assessed by electrospray mass spectrometry or by comparison of deconvoluted resonance intensity to the intensity of an internal standard of known concentration. For example, to produce >90% 6-19F-tryptophan–labeled E. coli dihydrofolate reductase, we used the E. coli auxotroph W3110TrpA33 containing the plasmid pTrc99DHFR. This plasmid was constructed by inserting the folA gene from plasmid pTY1 into the plasmid pTrc99A (Pharmacia Co., Piscataway, NJ). The cells were grown in a Biostat B fermentor (Braun Instruments, Allentown, PA) at 37 and pH 7 on M9 minimal medium supplemented with twice the normal concentration of phosphate salts and supplemented with 1.5 g/liter CSM-TRP (Bio-101 Inc., Vista, CA), 0.2% glucose, 0.2 mM l-tryptophan, 50 g/ml ampicillin, and 1 ml/ liter Poly-Vi-Sol vitamin drops with iron (Mead-Johnson, Evansville, IN). Cells were maintained at pH 7 by addition of NH4OH. Glucose was maintained between 0.1% and 0.2% and pO2 between 25% and 35% by varying the rate of addition of a feed mixture containing 45 g/liter CSM-TRP, 1.2 g/liter l-tryptophan, 30 g/liter NH4Cl, 4.8 g/liter MgSO4, and 20% glucose. The cells were harvested at A600 ¼ 16 and resuspended in fresh
29
I. J. Ropson and C. Frieden, Proc. Natl. Acad. Sci. USA 89, 7222 (1992).
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medium containing 0.2 mM 6-19F-tryptophan in place of l-tryptophan. After 30 min of growth in the 6-19F-tryptophan medium, the plasmid was induced with 1 mM isopropyl--d-thiogalactopyranoside (IPTG) for 2 h, and then cells were harvested. The yield is approximately 1 mg of protein per 1 g wet weight of cells. In the case of adenosine deaminase, bacteria (W3110TrpA33) containing the plasmid pQE80LmADA have been grown in Luria broth to A600 ¼ 4 and then harvested and washed twice with minimal media as described by Muchmore et al.30 containing 1 mM 6-19F-tryptophan. The plasmid was induced with 1 mM IPTG and grown for 3 h. Greater than 90% incorporation into adenosine deaminase was achieved (Shu and Frieden, unpublished observation). Initial use of rich media has the advantage of faster growth and possibly higher optical density than minimal media. To produce >90% labeled p-19F–labeled phenylalanine dihydrofolate reductase or E. coli PapD, we have used two phenylalanine auxotrophs, DL3930 and NK6024 (also called CGSC #6178).31 The former is auxotrophic for both phenylalanine and tyrosine. The latter has a transposon insertion encoding tetR in the pheA gene, which encodes the enzyme that converts chorismate to prephenate to phenylpyruvate. The bacteria are grown on media containing tetracycline to select for the insertion. Both strains grow well on minimal media as used above.30 We have achieved >90% labeling by growing DL39 containing the plasmid ptrc99DHFR or pQ80DHFR in the presence of 0.1 mM phenylalanine to an A600 of 3.0. At this point the cells have just stopped the logarithmic phase of growth. Cells are then harvested and resuspended in media containing 0.2 mM p-19F-phenylalanine. After 30 min, the plasmid is induced with 1 mM IPTG for 2 h and the bacteria are harvested. To produce p-19F-phenylalanine–labeled PapD we grow NK6024 containing the plasmid pQE80papD in minimal media containing 1 mM unlabeled phenylalanine and then harvest the cells while in log phase of growth (A600 ¼ 5). The cells are washed twice with 0.9% NaCl, 1 mM p-19F–labeled phenylalanine and then resuspended in new media containing 1 mM 19F-labeled amino acid. We have achieved >95% labeling (Bann and Frieden, unpublished observations) and this approach was outlined in the paper of Furter31 for the incorporation of p-19F–labeled phenylalanine in mouse dihydrofolate reductase.
30
D. C. Muchmore, L. P. Mclntosh, C. B. Russell, D. E. Anderson, and F. W. Dahlquist, Methods Enzymol. 177, 44 (1989). 31 R. Furter, Protein Sci. 7, 419 (1998).
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Assignment of Fluorine Resonances
As already mentioned, one drawback of fluorine NMR is a lack of a comprehensive theory to relate fluorine chemical shift to its environment as deduced, for example, from the known three-dimensional structure solved by X-ray crystallography or NMR spectroscopy. Therefore, the assignment of the fluorine resonances in a uniformly labeled protein is usually accomplished by site-directed mutagenesis. Each of the residues of the amino acid being uniformly labeled is mutated to another, which is chemically similar, such as a tyrosine to phenylalanine or tryptophan to phenylalanine. Each mutant protein is then fluorine labeled and purified using the procedure developed for the wild-type protein. 19F-NMR spectra of the mutant proteins are obtained under each of the conditions of interest (e.g., apo, in the presence of ligand or denatured). If the mutation has not introduced a significant structural perturbation, only one resonance will have disappeared from the spectrum, the remaining resonances will show minimal chemical shift perturbation, and the missing resonance will be assigned unambiguously to the mutated amino acid. A problem with this method is the potential to introduce some perturbations into the 19F-NMR spectra such that a remaining resonance undergoes a significant chemical shift change or disappears. Large spectral perturbations would probably be due to large changes in the structure or dynamics of the mutant protein by the chosen amino acid substitution. To minimize such effects, several tools are available. For example, one could search the sequence databases for homologous proteins using standard similarity search tools (i.e., BLAST via the internet athttp://us. expasy.org/tools/) to identify potential conservative substitutions. The BLOSUM62 scoring matrix32 quantifies the results of such an approach. If conservative substitutions cannot be identified using sequence search and alignment tools, or from a complementary approach, it may be useful to choose a substitution based on statistical analysis of a database of protein structures. Such methods attempt to identify ‘‘similar’’ amino acids based on structural elements (helix, sheet, turn etc.) or environment (solvent exposed vs. interior). An example of the former approach is PSIPRED33 accessible on the Internet via http://bioinf.cs.ucl.ac.uk/ psipred/. Examples of the latter approach can be found at http://prowl. rockefeller.edu/aainfo/contents.htm.
32 33
S. Henikoff and J. G. Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1992). D. T. Jones, J. Mol. Biol. 292, 195 (1999).
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Single-Site–Specific Labeling of Proteins
Labeling of a protein with a 19F-labeled amino acid at more than one site could affect the global stability of the protein. Thus, there is a potential for heterogeneity in the stabilizing/destabilizing effects of the fluorine substitution. An excellent example is the effect of a single fluorine substitution with different 19F-labeled tryptophan analogs in the protein annexin V.22 The X-ray crystal structures of wild-type, 4-, 5-, and 6-19F–labeled tryptophan annexin V were compared to the effects observed by circular dichroism on thermal stability. A decrease in thermal stability was observed for the 4- and 6-19F–labeled proteins, and this correlated with a decrease in molecular packing from the crystal structure, while the 5-19F-tryptophan showed no altered packing and a slight increase in thermal stability. Thus, even at a single site there can be heterogeneity in the effects on stability. In most cases, biosynthetic incorporation results in more than one site being labeled. Therefore, it would be highly advantageous to be able to selectively place a single 19F-labeled amino acid at a given position in the sequence in order to probe the effects on stability in that region only. This would be a minimal perturbation and thus most closely represent the native unlabeled protein. Furthermore, if the number of 19F-labeled amino acids is high, as in large proteins, or if a complex of proteins is studied, labeling at a single site would greatly simplify the observations. Furter31 recently developed such a system for the site-specific incorporation of p-19F–labeled phenylalanine, which relies upon the expression of three separate genes from two vectors: a yeast phenylalanyl tRNA synthetase, a yeast amber suppressor tRNA, and the gene of interest with an Amber mutation. The use of the heterologous yeast tRNA/synthetase pair is the cornerstone of this technique, since there is little cross-reactivity between the yeast tRNA/synthetase pair and the E. coli tRNA/synthetase pair. We have used this method to unambiguously assign the p-19F–labeled phenylalanine resonances of both PapD and dihydrofolate reductase from E. coli. The approach is simple and straightforward. An example is shown in Fig. 3 for PapD. Strains and Vectors The strain used for specific incorporation of a single phenylalanine is K10-F6. This is a p-19F-Phe–resistant, Phe auxotroph. There are two plasmids that are required for the ability to produce site-specifically labeled protein, and these have been outlined by Furter.31 One plasmid contains the yeast tRNA synthetase and the mouse DHFR gene (pRO148), and the other the tRNAPhe/amber (pRO117). Both plasmids and the strain
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Fig. 3. Site-specific labeling PapD with p-19F–labeled phenylalanine using the procedure described in Appendix 1. The data were collected at 20 using a fluorine cryoprobe on a Varian Unity-Plus 500-MHz spectrometer.
K10-F6 were obtained as gifts from Dr. David Tirrell (Caltech). The plasmid pRO148 is a derivative of pQE16, one of a set of vectors available from Qiagen that can allow expression of six-His–tagged coding sequences. pRO117 is a derivative of the pACYC177 plasmid pREP4, and also encodes the laclq gene. Both of the requisite genes (tRNA synthetase and tRNAPhe/amber from pRO148 and pRO117, respectively) can be subcloned after digestion with PvuII into blunt-end restriction sites, thus it should be feasible to utilize other vectors as long as they are compatible. Because overexpression of PapD is toxic to E. coli, it was essential to repress the papd gene by placing the laclq gene in cis rather than trans. Thus we utilized the pQE80 vector (Qiagen) that has this feature, but also has a six-His coding sequence between the start codon and the multiple cloning site. To remove the six-His tag sequence we used polymerase chain reaction (PCR) to generate a fragment that could be incorporated into the EcoR1 site upstream of the RBS and ATG start sites, and a KpnI site within the multicloning site of pQE80. Thus, the forward primer should include the RBS and ATG start sites that are normally present in the pQE80 plasmid, with an additional 12 bases of coding sequence from the gene of interest. For instance, to remove the six-His tag and introduce the papd gene into pQE80, we used as the forward primer the sequence CCCGAATTCATTAAAGAGGAGAAATTAACTATGATTCGAAAAAAG. This primer encoded the pQE80 EcoR1 site, the RBS site, and the ATG start site. If the gene of interest is not toxic, or if the six-His tag is
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cooperativity in protein folding and assembly
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desired for ease of purification, then the gene of interest can be subcloned using the multicloning sites provided in the appropriate vector. Fluorine-Labeled Amino Acids That Can Be Incorporated into Proteins
As implied above, there are a number of fluorine-labeled amino acids commercially available. These include the 4-, 5-, and 6-19F-tryptophan, the o-, m-, and p-19F-phenylalanine, m-19F-phenylalanine, m-19F-tyrosine, and 19F-methylhistidine. Incorporation of difluoromethionine,34 hexafluoroleucine,35a (2S,4R)-5-19F-leucine,35b and 19F-proline35c has been reported. These other analogs will provide a valuable tool for the investigation of protein structure and folding. The recent discovery of a fluorinase36 may allow other fluorine-labeled amino acids to be synthesized enzymatically. Currently only 19F-phenylalanine can be incorporated site specifically as described above. On the other hand, Wang et al.37 reported a method for site-specific incorporation of O-methyl tyrosine suggesting that it may be possible to incorporate 19F-tyrosine site specifically. The chapter by Gerig5 includes a number of suggestions for the incorporation of fluorine labels into proteins. The reader would be well served by consulting this reference. Site-Specific Labeling with Cysteine-Reactive Compounds
Another method that has been used for site specifically labeling proteins with fluorine for 19F-NMR experiments is the use of compounds that are reactive to cysteine residues. This approach was pioneered by Gerig5 and has the advantage that one can probe a single site. Obviously, the effect of the cysteine mutation, before and after treatment with a fluorinated compound, must be taken into account. However, a major advantage of cysteine labeling is that one can use these sites to probe with other cysteine-reactive reagents, such as fluorescent or spin labels, for comparative purposes. A recent study of the integral membrane protein rhodopsin has shown that fluorine labeling of two cysteine residues that are close in space can allow one to obtain through-space information under different conditions (i.e., light and dark) using 19F nuclear Overhauser effects (NOEs).38 34
M. D. Vaughn, P. Cleve, V. Robinson, H. S. Duewel, and J. F. Honek, J. Am. Chem. Soc. 121, 8475 (1999). 35 (a) Y. Tang and D. A. Tirrell, J. Am. Chem. Soc. 123, 11089 (2001). (b) J. Feeney et al., J. Am. Chem. Soc. 118, 8700 (1996). (c) C. Renner et al., Angew Chem. Int. Ed. 40, 923 (2001). 36 D. O’Hagan, C. Schaffrath, S. L. Cobb, J. T. Hamilton, and C. D. Murphy, Nature 416, 279 (2002). 37 L. Wang, A. Brock, B. Herberich, and P. G. Schultz, Science 292, 498 (2001). 38 M. C. Loewen et al., Proc. Natl. Acad. Sci. USA 98, 4888 (2001).
19
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TABLE II Cysteine-Reactive Fluorinating Reagents Formula
Name; Commercial availability
Reference
CF3-CH2-SH CF3-CO-CH2-Br F-C4H4-SH CF3-C4H4-NH-CO-CH2l (CF3)3C-C4H4-NH-CO-CH2l
2,2,2-Trifluoroethanethiol; yes 3-Bromo-1,1,1-trifluoroacetone; yes 4-Fluorobenzenethiol; yes 4-(Trifluoromethyl)phenyliodoacetamide; no 4-(Perfluoro-tert-(butyl)phenyliodoacetamide; no
38, 39 41, 42 43 44 45
There are a number of different compounds that can be used to label cysteines, and some recently used ones are presented in Table II. Although several cysteine-reactive fluorocompounds have been used recently for 19 F-NMR studies, a highly selective reagent for cysteine is the use of trifluoroethylthiol (TET).38,39 Whereas a fluorohalogenated compound may in some cases show reactivity toward amines, TET labeling of cysteine is specific for the formation of a disulfide. TET labeling produces an analog that is similar in length to lysine and chemically similar to methionine, such that either substitution [Lys to Cys (TET) or Met to Cys (TET)] would probably result in minimal perturbation of the molecule. Also, this compound exhibits a sharp resonance, due to the lack of proton coupling of the CF3 group.40–45 Conclusions
This chapter outlines the methods that can be used to incorporate fluorine-labeled amino acids, as well as other ligands, into proteins. There are many reasons for using these techniques to study the structural and dynamic aspects of proteins. Of these the most dramatic is the sensitivity of the fluorine nucleus to its environment under conditions in which there are only minimal perturbations in the wild-type structure. Thus, studies can investigate the changes due to ligand binding, protein–protein interactions, as well as protein folding. Currently no good theory exists for 39
J. Klein-Seetharaman, E. V. Getmanova, M. C. Loewen, P. J. Reeves, and H. G. Khorana, Proc. Natl. Acad. Sci. USA 96, 13744 (1999). 40 G. R. Drapeau, W. J. Brammar, and C. Yanofsky, J. Mol. Biol. 35, 357 (1968). 41 M. R. Thomas and S. G. Boxer, Biochemistry 40, 8588 (2001). 42 K. Oxenoid, F. D. Sonnichsen, and C. R. Sanders, Biochemistry 41, 12876 (2002). 43 J. P. Caradonna, E. W. Harlan, and R. H. Holm, J. Am. Chem. Soc. 108, 7856 (1986). 44 J. W. Shriver and B. D. Sykes, Biochemistry 21, 3022 (1982). 45 D. Heintz, H. Kany, and H. R. Kalbitzer, Biochemistry 35, 12686 (1996).
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relating chemical shifts to the structural environment. When such a theory is developed, the amount of information that can be obtained from fluorine chemical shifts should be impressive. Appendix I: General Method for the Production of Proteins Site Specifically Labeled with p-19F-Phenylalanine Using PapD as an Example
Originally, the procedure described by Furter for labeling mouse dihydrofolate reductase was to grow the bacteria in M9 minimal media supplemented with phenylalanine/p-19F-phenylalanine (0.2 and 3 mM, respectively).31 This serves the purpose of keeping a selective pressure to maintain the resistance to p-19F–labeled phenyalanine. In the original paper,31 the cells were grown to an A600 of 1.0 and then shifted to media containing 3 mM p-19F-phenylalanine and 0.03 or 0.04 mM phenylalanine. It was found that the level of specific p-19F-phenylalanine incorporation increased with ‘‘an unproportional 2.3-fold increase in p-19F-phenylalanine contamination’’ when using 0.03 versus 0.04 mM phenylalanine. Because the level of specific incorporation is important, we use the 0.04 mM phenylalanine final concentration for labeling, and find very similar levels of specific incorporation over uniformly labeled protein. A modified protocol for site-specific labeling is described below and should be applicable to other proteins. Two days prior to the growth of bacteria, frozen K10F6 cells harboring the pQE80papDPhe!Amber mutant/pRO117 are streaked onto LB agar plates containing 100 g/ml ampicillin and 50 g/ml kanamycin. A single colony is picked and grown the morning of the following day in 5 ml of LB containing antibiotics at 37 . In the evening, the cells are diluted 1:1000 into 100 ml of the defined media containing 0.2 mM Phe/3 mM p-19F-phenylalanine with antibiotics. The defined media we use30 contains (for a 1-liter volume) 0.2 mM phenylalanine/3 mM p-19F-phenylalanine, 0.5 g alanine, 0.4 g arginine, 0.4 g aspartate, 0.05 g cystine, 0.4 g glutamine, 0.65 g glutamate, 0.55 g glycine, 0.1 g histidine, 0.23 g isoleucine, 0.23 g leucine, 0.42 g lysine hydrochloride, 0.25 g methionine, 0.1 g proline, 2.1 g serine, 0.23 g threonine, 0.17 g tyrosine, 0.23 g valine, 0.5 g adenine, 0.65 g guanosine, 0.2 g thymine, 0.5 g uracil, 0.2 g cytosine, 1.5 g sodium acetate, 1.5 g succinic acid, 0.5 g NH4Cl, 0.85 g NaOH, and 10.5 g K2PO4 per 950 ml of H2O. This is then autoclaved. To this is added 50 ml of a sterile-filtered solution of 40% glucose, 4 ml of 1 M MgSO4, and 10 ml of a sterile solution of 2.7 mg FeCl36H2O, 2 mg CaCl22H2O, 2 mg ZnSO47H2O, 2 mg MnSO4H2O, 50 mg l-tryptophan, 50 mg thiamine, 50 mg niacin, and 1 mg biotin. This solution has a pH
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near 7.2. The antibiotics (1 ml of 50 mg/ml kanamycin, 1 ml of 100 mg/ml ampicillin) are added separately. We have found that it is important to maintain the bacteria in a logarithmic phase of growth and not to let them go into stationary phase, as we have observed premature lysis of the bacteria. Thus, the overnight culture is started rather late (7:00 pm). The bacteria from the overnight culture are then diluted 1:50 into 750 ml of media in Fernbach flasks. The cells are grown to an A600 of 1.0, and harvested by centrifugation. Once harvested, the cells are then washed twice with 0.9% NaCl, 3 mM p-19F-phenylalanine. The resuspended cells are grown for about 20–30 min. min. Then, IPTG is added to a final concentration of 1.0 mM, and the cells are grown for an additional 1–3 h. Following this, the usual procedures for purifying the protein of choice are applicable.
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natural BPTI, when water soluble, form conformational ensembles that favor nativelike structure. To produce a native state mimic, we incorporate two or more core modules into larger peptides in which module–module interactions are promoted. The interactions are expected to be mutually stabilizing, and thus to further shift the conformational bias of component core modules toward more ordered structure. This idea has been realized in the protein BetaCore, in which two core modules are incorporated into a single molecule by means of a long cross-link.36,37 BetaCore is monomeric in water and forms a new fold composed of a four-stranded, antiparallel -sheet. The single, dominant conformation of BetaCore has been characterized by various NMR experiments. Acknowledgments We thank Andrew Robertson and D. Wayne Bolen for critical reading of the manuscript and helpful discussions. This work is currently supported by NIH Grant GM51628. 36 37
N. Carulla, C. Woodward, and G. Barany, Protein Sci. 11, 1539 (2002). N. Carulla, G. Barany, and C. Woodward, Biophys. Chem. 101–102, 67 (2002).
[18] The Preparation of 19F-Labeled Proteins for NMR Studies By Carl Frieden, Sydney D. Hoeltzli, and James G. Bann Introduction
The incorporation of 19F-labeled amino acids into proteins for nuclear magnetic resonance (NMR) spectroscopy has been a technique used for many years as a probe of protein structure and dynamics. Three previous articles in Methods of Enzymology1–3 have dealt with this subject. Two recent excellent reviews4,5 describe the advantages of using 19F-NMR for such studies and cover the field well, pointing out the usefulness of fluorine as a probe of local environment. The present chapter discusses methods of incorporating 19F-labeled amino acids into proteins. 1
B. D. Sykes and W. E. Hull, Methods Enzymol. 49, 271 (1978). G. Horton and I. Boime, Methods Enzymol. 96, 777 (1983). 3 J. T. Gerig, Methods Enzymol. 177, 3 (1989). 4 M. A. Danielson and J. J. Falke, Annu. Rev. Biophys. Biomol. Struct. 25, 163 (1996). 5 J. T. Gerig, in ‘‘Biophysical Textbook Online’’ (2001). www.biophys.org/btol/ 2
METHODS IN ENZYMOLOGY, VOL. 380
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As noted in the two recent reviews,4,5 there are many properties of fluorine that make it a useful probe for studies of protein structure and function. For example, the fluorine nucleus is small, only slightly larger than the hydrogen nucleus. Although the dipole moment is considerably different, it is expected that there will be a minimal perturbation to the structure, stability, and functionality of the protein. Fluorine does not occur naturally in proteins so that a protein labeled with an 19F-labeled amino acid will exhibit NMR peaks due only to the label. Because fluorine is extraordinarily sensitive to its environment, and to local shielding effects,6 the NMR peaks are typically well resolved from one another in a one-dimensional (1D) NMR spectrum. This sensitivity makes 19 F-NMR extremely well suited for studies of both protein structure and protein folding, since even in the denatured state the peaks are frequently resolved.7–10 It should also be noted that 19F-NMR can be used to examine structural aspects of much higher molecular weight protein than are generally accessible by proton NMR. Under conditions in which two or more conformers exist in equilibrium, it may be possible to use 19F-NMR to calculate rate or dissociation constants if the conformers have different chemical shifts. If, for example, two different conformers exchange slowly on the NMR time scale, there will be two peaks and dissociation constants can be determined by quantifying the area of each peak. For conformers or liganded forms in intermediate exchange on the NMR time scale (roughly 5–5000 s1 depending upon chemical shift difference), the exchange rate kex may be determined by lineshape analysis.11 For conformers or liganded forms in slow exchange, in the millisecond range, the exchange rate may be accessible through two-dimensional experiments such as nuclear Overhauser enhancement spectroscopy (NOESY). Finally, the accessibility of a residue to bound or bulk water can be assessed by 19F-1H HOESY experiments.12 When using 19F-NMR to study protein folding, two major types of experiments can be carried out: an equilibrium measurement that involves the determination of peak chemical shift and intensity as a function of denaturant concentration or a kinetic experiment measuring peak intensity as a function of time after dilution of the denaturant. This latter can be done by manual mixing or, for faster reactions (1 s or longer), using a 6
E. Y. Lau and J. T. Gerig, Biophys. J. 73, 1579 (1997). S. D. Hoeltzli and C. Frieden, Biochemistry 33, 5502 (1994). 8 S. D. Hoeltzli and C. Frieden, Biochemistry 35, 16843 (1996). 9 S. D. Hoeltzli and C. Frieden, Biochemistry 37, 387 (1998). 10 J. G. Bann, J. Pinkner, S. J. Hultgren, and C. Frieden, Proc. Natl. Acad. Sci. USA 99, 709 (2002). 11 J. Sandstrom, ‘‘Dynamic NMR Spectroscopy.’’ Academic Press, New York, 1982. 12 D. P. Cistola and K. B. Hall, J. Biomol. NMR 5, 415 (1995). 7
402
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Fig. 1. 19F-NMR spectra of apo, binary, and ternary complexes of E. coli dihydrofolate reductase labeled with 6-19F-tryptophan. The data were collected at 22 on a Varian VXR 500 equipped with a Nalorac indirect detection probe. The five resonances were assigned using site-directed mutagenesis. MTX is the dihydrofolate analog, methotrexate. Data taken from Hoeltzli and Frieden.7
stopped-flow device.13 Because unfolded chemical shifts are frequently resolved, it is also possible to assess each residue individually in the unfolded state. Note that these measurements are different from those of hydrogen/ deuterium exchange in that the latter measures the ability of backbone amide protons to exchange with the solvent while the 19F-NMR experiments assess the environment of a specific amino acid side chain. Figure 1 illustrates a typical equilibrium experiment with native Escherichia coli dihydrofolate reductase. In this figure, the five tryptophan residues have been substituted with 6-19F-tryptophan. There are five clearly separated peaks for the native protein. Addition of ligands such as NADP or methotrexate (MTX) affects some peaks more dramatically than others. Four peaks are observed in the denatured protein (data not shown). In the presence of urea, all peaks in the native protein, except for Trp-22, are in slow exchange with the denatured form. In a technological advance, a fluorine cryoprobe (Varian) is currently available allowing either fewer transients or lower protein concentrations to be used for a given signal-to-noise (S/N) ratio. In our experience the S/N ratio is at least 4-to 5-fold greater than the conventional probes currently in use. This is particularly important for studies of proteins that tend 13
S. D. Hoeltzli, I. J. Ropson, and C. Frieden, in ‘‘Techniques in Protein Chemistry,’’ pp. 455–465. Academic Press, New York, 1994.
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Fig. 2. 19F-NMR spectra of 6-19F-tryptophan–labeled PapD recorded as a function of time after a stopped-flow urea jump from 4.5 M urea to 2.25 M urea. The final concentration of protein was 70 M using a fluorine cryoprobe on a Varian Unity-Plus 500-MHz spectrometer with 32 transients at each time point. Data were obtained at 20 as described by Bann et al.10 The buffer was 30 mM Mops/HCl, pH 7.0.
to aggregate at higher concentrations, or for detecting peaks that may have previously been difficult to quantify. Figure 2 shows a kinetic experiment using 70 M of the protein PapD and the fluorine cryoprobe. PapD, a bacterial chaperone, is a two-domain protein in which each domain has one tryptophan. In these experiments we used stopped-flow methods as described elsewhere.7–10 As shown by Fig. 2, changes in peak intensity can be measured as a function of time after dilution of the denaturant. Interestingly, an intermediate form, represented by the peak at 45.5 ppm, is present during refolding and disappears as the protein finishes the folding process. Although not shown in Fig. 2, it should be pointed out that the earliest time one can collect a spectrum after diluting out the denaturant is about 1–2 s. Unfortunately, there is a least one problem that has not been solved: it has not been easy, so far, to relate the chemical shift of the fluorine signal to the structural environment surrounding the fluorine within the protein.5,14 Therefore, it is not yet possible to predict the chemical shifts in a 19 F-NMR spectrum based on analysis of the protein’s amino acid sequence or the analysis of its three-dimensional structure. Consequently, to assign 14
J. G. Pearson et al., Biochemistry 36, 3590 (1997).
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the peaks from the labeled amino acids in a spectrum, one needs to use sitedirected mutagenesis. Techniques used for this method will be outlined in detail in this chapter. Currently, there are a number of different analogs that can be used to label proteins biosynthetically, and the list of available amino acids continues to grow as improved methods for the efficient syntheses of these amino acids continue.15 Most studies thus far have utilized aromatic amino acids for reasons discussed later. Incorporation of
19
F-Labeled Amino Acids into Proteins
For production of proteins containing 19F-labeled amino acids, biosynthetic incorporation of the labeled amino acids by microbial protein expression is the strategy of choice. High yields of >90% labeled protein are possible and biosynthetic methods are cost effective relative to chemical or in vitro synthesis. High levels of incorporation are important since this allows lower protein concentrations to be used and avoids a heterogeneous population of labeled protein. A number of approaches have been successfully utilized to produce very high label incorporation and two common themes emerge: (1) the importance of placing the gene for the protein of interest under control of a tightly regulated inducible promoter and (2) the critical issue of completely depleting and preventing synthesis of, natural amino acids from the labeling media prior to inducing protein production. Because 19F-labeled amino acids can inhibit bacterial growth to differing degrees16 the bacteria usually cannot be grown from inoculation on fluorine-containing medium. Bacterial cultures can be grown on defined medium containing a limited amount of the unlabeled amino acid and then either grown to the point where unlabeled amino acid is completely depleted prior to introduction of the 19F-labeled amino acid (for example, Kranz et al.17) or harvested and transferred to defined medium containing 19 F-labeled amino acid. Our laboratory has obtained more consistent results for a number of different proteins with the latter approach. It should be noted that some proteins cannot be produced under conditions that allow complete labeling (for example, Luck and Falke18), and in these cases it may be necessary to accept a lower level of fluorine label incorporation by adding unlabeled amino acid to the labeling medium or by incompletely depleting the unlabeled amino acid. 15
A. Sutherland and C. L. Willis, Nat. Prod. Rep. 17, 621 (2000). R. E. Marquis, in ‘‘Handbook of Experimental Pharmacology’’ (F. A. Smith, ed.), Vol. 20. pp. 165–192. Springer-Verlag, Berlin, 1970. 17 J. K. Kranz, J. Lu, and K. B. Hall, Protein Sci. 5, 1567 (1996). 18 L. A. Luck and J. J. Falke, Biochemistry 30, 4257 (1991). 16
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Labeling with Aromatic Amino Acids To date, most studies of 19F-labeled proteins have involved incorporation of 19F-labeled aromatic amino acids. There are several reasons for this. Usually, even quite large proteins contain a limited number of aromatic amino acids, simplifying the problem of assignment. Several healthy strains of bacteria auxotrophic for each of the aromatic amino acids are readily available. In addition, m-19F-tyrosine, 4-, 5-, and 6-19F–labeled tryptophan, and m-, o-, and p-19F-phenylalanine are all commercially available at reasonable cost. All of these analogs have been used sucessfully in studies of various proteins. In some studies, more than one analog has been successfully incorporated into the same protein.19–22 These results and those from our laboratory (Ropson and Frieden, unpublished observations; Hoeltzli and Frieden, unpublished observations) suggest that proteins labeled with different analogs can express at different levels, can differ in chemical shift resolution, and might differ in structural perturbation and stability. Examination of the crystal structure and the local contacts that are likely to be affected by the substitution will certainly help guide the experimentalist in the choice of an analog. It may be necessary to first try a series of analogs to determine if there is an effect on stability. In the absence of a comprehensive theory predicting fluorine chemical shifts the choice of analog remains largely empirical. Use of Nonauxotrophic Strains. The simplest approach to incorporation of a 19F-labeled amino acid is to use whatever nonauxotrophic bacterial strain has been found to give good protein expression and to repress endogenous amino acid synthesis with high concentrations of amino acids. This approach is limited by the fact that 19F-labeled amino acids are generally inhibitory to bacterial growth. However, Lu and co-workers23 achieved incorporation of greater than 90% 3-19F-tyrosine into lac repressor protein by using high concentrations of tryptophan, phenylalanine, and tyrosine to repress 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase, the first enzyme of the aromatic pathway. Strain CSH46 (also known as 96) was grown in M9 minimal media supplemented with 1% glucose, 1 g/ml thiamine, and 0.2 mM amino acids except tyrosine, tryptophan, and phenylalanine. Tryptophan and phenylalanine were supplemented at 1 mM, tyrosine was omitted, and the cells grown to A550 nm of 1.0. The cultures 19
G. S. Rule, E. A. Pratt, V. Simplaceanu, and C. Ho, Biochemistry 26, 549 (1987). C. Lian et al., Biochemistry 33, 5238 (1994). 21 M. A. Dominguez, Jr., K. C. Thornton, M. G. Melendez, and C. M. Dupureur, Proteins 45, 55 (2001). 22 C. Minks, R. Huber, L. Moroder, and N. Budisa, Biochemistry 38, 10649 (1999). 23 P. Lu, M. Jarema, K. Mosser, and W. E. Daniel, Proc. Natl. Acad. Sci. USA 73, 3471 (1976). 20
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were shifted to 42 to induce production of lac repressor and 1 mM 3-19F-tyrosine added. Use of a nonauxotrophic strain to incorporate 19 F-labeled amino acids has resulted in lower degrees of label incorporation in other reports.21,24 Careful attention to optimizing the inhibition of amino acid synthesis and the concentration of labeled amino acid is probably critical to success. Whether or not high (>90%) levels of label incorporation can be obtained, this approach may be valuable when expression of the protein of interest requires specific properties not available in an existing auxotrophic strain. A second approach is the use of glyphosate to induce aromatic amino acid auxotrophy in a nonauxotrophic bacterial strain. Glyphosate is an inhibitor of 5-enolpyruvylshikimic acid 3-phosphate biosynthesis and suppresses the production of aromatic metabolites, including the aromatic amino acids tryptophan, tyrosine, and phenylalanine.25 This strategy may be especially useful when an amino acid auxotroph of desirable properties is not available, or when the protein of interest cannot be expressed in available auxotrophs.26 Recently, complete 5-fluorotryptophan labeling of T. maritima cold shock protein using glyphosate to repress aromatic amino acid synthesis has been reported by Schuler et al.27 Briefly, cells were grown at 37 in minimal medium containing 50 mg/liter of all amino acids except tryptophan. Aromatic amino acid synthesis was suppressed with 1 g/liter glyphosate. When the cultures left exponential growth at A550 nm 1.5, 50 mg/liter dl-5-19F-tryptophan was added and protein expression induced for 4 h prior to harvest. Use of glyphosate has resulted in lower levels of incorporation in other reports.28 Depletion of the unlabeled amino acid from the growth medium is critical and needs to be monitored carefully. Use of Auxotrophic Strains. Most studies of 19F-labeled amino acids incorporated into proteins have utilized bacterial strains auxotrophic for the amino acid of interest. Numerous aromatic amino acid auxotrophs are available from individual investigators, through the ATCC or the Yale-New Haven E. coli Genetic Stock Center. The properties and sources of several auxotrophs we have found to be particularly useful are listed in Table I. Generally, the auxotrophic bacteria are transformed with a plasmid encoding the gene for the protein of interest under the control of a tightly regulated promoter using standard techniques. Transformed bacteria are grown on rich or defined medium containing a specific concentration of 24
C. M. Dupureur and L. M. Hallman, Eur. J. Biochem. 261, 261 (1999). L. Comai, L. C. Sen, and D. M. Stalker, Science 221, 370 (1983). 26 P. Bai, L. Luo, and Z. Peng, Biochemistry 39, 372 (2000). 27 B. Schuler, W. Kremer, H. R. Kalbitzer, and R. Jaenicke, Biochemistry 41, 11670 (2002). 28 H. W. Kim, J. A. Perez, S. J. Ferguson, and I. D. Campbell, FEBS Lett. 272, 34 (1990). 25
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TABLE I Useful E. COLI Auxotrophsa Strain
Auxotrophy
Mutation
Reference
W3110TrpA33 DL39 (CGSC #6913) NK6024 (CGSC #6178)
Trp Asp, Ile, Leu, Phe, Tyr Phe
trpA33 tyrB pheA::Tn10 (tetR)
40 30 31
a
Strains are available from the E. coli genetic stock center (http://cgsc.biology.yale.edu).
the natural amino acid of interest, then harvested and resuspended in defined medium containing the fluorine-labeled amino acid. The cells are allowed to recover for a period of time sufficient to deplete intracellular stores of unlabeled amino acid and protein production is then induced for an optimal period of time and cells harvested. Using this method, we have successfully produced 6-19F-tryptophan–labeled rat intestinal fatty acid binding protein,29 6-19F-tryptophan8,9 and p-19F-phenylalanine–labeled E. coli dihydrofolate reductase (Hoeltzli and Frieden, unpublished observation), 6-19F-tryptophan10 and p-19F-phenylalanine–labeled E. coli PapD (Bann and Frieden, unpublished data), and 6-19F-tryptophan–labeled murine adenosine deaminase (Shu and Frieden, unpublished data), with greater than 90% incorporation of label as assessed by electrospray mass spectrometry or by comparison of deconvoluted resonance intensity to the intensity of an internal standard of known concentration. For example, to produce >90% 6-19F-tryptophan–labeled E. coli dihydrofolate reductase, we used the E. coli auxotroph W3110TrpA33 containing the plasmid pTrc99DHFR. This plasmid was constructed by inserting the folA gene from plasmid pTY1 into the plasmid pTrc99A (Pharmacia Co., Piscataway, NJ). The cells were grown in a Biostat B fermentor (Braun Instruments, Allentown, PA) at 37 and pH 7 on M9 minimal medium supplemented with twice the normal concentration of phosphate salts and supplemented with 1.5 g/liter CSM-TRP (Bio-101 Inc., Vista, CA), 0.2% glucose, 0.2 mM l-tryptophan, 50 g/ml ampicillin, and 1 ml/ liter Poly-Vi-Sol vitamin drops with iron (Mead-Johnson, Evansville, IN). Cells were maintained at pH 7 by addition of NH4OH. Glucose was maintained between 0.1% and 0.2% and pO2 between 25% and 35% by varying the rate of addition of a feed mixture containing 45 g/liter CSM-TRP, 1.2 g/liter l-tryptophan, 30 g/liter NH4Cl, 4.8 g/liter MgSO4, and 20% glucose. The cells were harvested at A600 ¼ 16 and resuspended in fresh
29
I. J. Ropson and C. Frieden, Proc. Natl. Acad. Sci. USA 89, 7222 (1992).
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medium containing 0.2 mM 6-19F-tryptophan in place of l-tryptophan. After 30 min of growth in the 6-19F-tryptophan medium, the plasmid was induced with 1 mM isopropyl--d-thiogalactopyranoside (IPTG) for 2 h, and then cells were harvested. The yield is approximately 1 mg of protein per 1 g wet weight of cells. In the case of adenosine deaminase, bacteria (W3110TrpA33) containing the plasmid pQE80LmADA have been grown in Luria broth to A600 ¼ 4 and then harvested and washed twice with minimal media as described by Muchmore et al.30 containing 1 mM 6-19F-tryptophan. The plasmid was induced with 1 mM IPTG and grown for 3 h. Greater than 90% incorporation into adenosine deaminase was achieved (Shu and Frieden, unpublished observation). Initial use of rich media has the advantage of faster growth and possibly higher optical density than minimal media. To produce >90% labeled p-19F–labeled phenylalanine dihydrofolate reductase or E. coli PapD, we have used two phenylalanine auxotrophs, DL3930 and NK6024 (also called CGSC #6178).31 The former is auxotrophic for both phenylalanine and tyrosine. The latter has a transposon insertion encoding tetR in the pheA gene, which encodes the enzyme that converts chorismate to prephenate to phenylpyruvate. The bacteria are grown on media containing tetracycline to select for the insertion. Both strains grow well on minimal media as used above.30 We have achieved >90% labeling by growing DL39 containing the plasmid ptrc99DHFR or pQ80DHFR in the presence of 0.1 mM phenylalanine to an A600 of 3.0. At this point the cells have just stopped the logarithmic phase of growth. Cells are then harvested and resuspended in media containing 0.2 mM p-19F-phenylalanine. After 30 min, the plasmid is induced with 1 mM IPTG for 2 h and the bacteria are harvested. To produce p-19F-phenylalanine–labeled PapD we grow NK6024 containing the plasmid pQE80papD in minimal media containing 1 mM unlabeled phenylalanine and then harvest the cells while in log phase of growth (A600 ¼ 5). The cells are washed twice with 0.9% NaCl, 1 mM p-19F–labeled phenylalanine and then resuspended in new media containing 1 mM 19F-labeled amino acid. We have achieved >95% labeling (Bann and Frieden, unpublished observations) and this approach was outlined in the paper of Furter31 for the incorporation of p-19F–labeled phenylalanine in mouse dihydrofolate reductase.
30
D. C. Muchmore, L. P. Mclntosh, C. B. Russell, D. E. Anderson, and F. W. Dahlquist, Methods Enzymol. 177, 44 (1989). 31 R. Furter, Protein Sci. 7, 419 (1998).
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Assignment of Fluorine Resonances
As already mentioned, one drawback of fluorine NMR is a lack of a comprehensive theory to relate fluorine chemical shift to its environment as deduced, for example, from the known three-dimensional structure solved by X-ray crystallography or NMR spectroscopy. Therefore, the assignment of the fluorine resonances in a uniformly labeled protein is usually accomplished by site-directed mutagenesis. Each of the residues of the amino acid being uniformly labeled is mutated to another, which is chemically similar, such as a tyrosine to phenylalanine or tryptophan to phenylalanine. Each mutant protein is then fluorine labeled and purified using the procedure developed for the wild-type protein. 19F-NMR spectra of the mutant proteins are obtained under each of the conditions of interest (e.g., apo, in the presence of ligand or denatured). If the mutation has not introduced a significant structural perturbation, only one resonance will have disappeared from the spectrum, the remaining resonances will show minimal chemical shift perturbation, and the missing resonance will be assigned unambiguously to the mutated amino acid. A problem with this method is the potential to introduce some perturbations into the 19F-NMR spectra such that a remaining resonance undergoes a significant chemical shift change or disappears. Large spectral perturbations would probably be due to large changes in the structure or dynamics of the mutant protein by the chosen amino acid substitution. To minimize such effects, several tools are available. For example, one could search the sequence databases for homologous proteins using standard similarity search tools (i.e., BLAST via the internet athttp://us. expasy.org/tools/) to identify potential conservative substitutions. The BLOSUM62 scoring matrix32 quantifies the results of such an approach. If conservative substitutions cannot be identified using sequence search and alignment tools, or from a complementary approach, it may be useful to choose a substitution based on statistical analysis of a database of protein structures. Such methods attempt to identify ‘‘similar’’ amino acids based on structural elements (helix, sheet, turn etc.) or environment (solvent exposed vs. interior). An example of the former approach is PSIPRED33 accessible on the Internet via http://bioinf.cs.ucl.ac.uk/ psipred/. Examples of the latter approach can be found at http://prowl. rockefeller.edu/aainfo/contents.htm.
32 33
S. Henikoff and J. G. Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1992). D. T. Jones, J. Mol. Biol. 292, 195 (1999).
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Single-Site–Specific Labeling of Proteins
Labeling of a protein with a 19F-labeled amino acid at more than one site could affect the global stability of the protein. Thus, there is a potential for heterogeneity in the stabilizing/destabilizing effects of the fluorine substitution. An excellent example is the effect of a single fluorine substitution with different 19F-labeled tryptophan analogs in the protein annexin V.22 The X-ray crystal structures of wild-type, 4-, 5-, and 6-19F–labeled tryptophan annexin V were compared to the effects observed by circular dichroism on thermal stability. A decrease in thermal stability was observed for the 4- and 6-19F–labeled proteins, and this correlated with a decrease in molecular packing from the crystal structure, while the 5-19F-tryptophan showed no altered packing and a slight increase in thermal stability. Thus, even at a single site there can be heterogeneity in the effects on stability. In most cases, biosynthetic incorporation results in more than one site being labeled. Therefore, it would be highly advantageous to be able to selectively place a single 19F-labeled amino acid at a given position in the sequence in order to probe the effects on stability in that region only. This would be a minimal perturbation and thus most closely represent the native unlabeled protein. Furthermore, if the number of 19F-labeled amino acids is high, as in large proteins, or if a complex of proteins is studied, labeling at a single site would greatly simplify the observations. Furter31 recently developed such a system for the site-specific incorporation of p-19F–labeled phenylalanine, which relies upon the expression of three separate genes from two vectors: a yeast phenylalanyl tRNA synthetase, a yeast amber suppressor tRNA, and the gene of interest with an Amber mutation. The use of the heterologous yeast tRNA/synthetase pair is the cornerstone of this technique, since there is little cross-reactivity between the yeast tRNA/synthetase pair and the E. coli tRNA/synthetase pair. We have used this method to unambiguously assign the p-19F–labeled phenylalanine resonances of both PapD and dihydrofolate reductase from E. coli. The approach is simple and straightforward. An example is shown in Fig. 3 for PapD. Strains and Vectors The strain used for specific incorporation of a single phenylalanine is K10-F6. This is a p-19F-Phe–resistant, Phe auxotroph. There are two plasmids that are required for the ability to produce site-specifically labeled protein, and these have been outlined by Furter.31 One plasmid contains the yeast tRNA synthetase and the mouse DHFR gene (pRO148), and the other the tRNAPhe/amber (pRO117). Both plasmids and the strain
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Fig. 3. Site-specific labeling PapD with p-19F–labeled phenylalanine using the procedure described in Appendix 1. The data were collected at 20 using a fluorine cryoprobe on a Varian Unity-Plus 500-MHz spectrometer.
K10-F6 were obtained as gifts from Dr. David Tirrell (Caltech). The plasmid pRO148 is a derivative of pQE16, one of a set of vectors available from Qiagen that can allow expression of six-His–tagged coding sequences. pRO117 is a derivative of the pACYC177 plasmid pREP4, and also encodes the laclq gene. Both of the requisite genes (tRNA synthetase and tRNAPhe/amber from pRO148 and pRO117, respectively) can be subcloned after digestion with PvuII into blunt-end restriction sites, thus it should be feasible to utilize other vectors as long as they are compatible. Because overexpression of PapD is toxic to E. coli, it was essential to repress the papd gene by placing the laclq gene in cis rather than trans. Thus we utilized the pQE80 vector (Qiagen) that has this feature, but also has a six-His coding sequence between the start codon and the multiple cloning site. To remove the six-His tag sequence we used polymerase chain reaction (PCR) to generate a fragment that could be incorporated into the EcoR1 site upstream of the RBS and ATG start sites, and a KpnI site within the multicloning site of pQE80. Thus, the forward primer should include the RBS and ATG start sites that are normally present in the pQE80 plasmid, with an additional 12 bases of coding sequence from the gene of interest. For instance, to remove the six-His tag and introduce the papd gene into pQE80, we used as the forward primer the sequence CCCGAATTCATTAAAGAGGAGAAATTAACTATGATTCGAAAAAAG. This primer encoded the pQE80 EcoR1 site, the RBS site, and the ATG start site. If the gene of interest is not toxic, or if the six-His tag is
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desired for ease of purification, then the gene of interest can be subcloned using the multicloning sites provided in the appropriate vector. Fluorine-Labeled Amino Acids That Can Be Incorporated into Proteins
As implied above, there are a number of fluorine-labeled amino acids commercially available. These include the 4-, 5-, and 6-19F-tryptophan, the o-, m-, and p-19F-phenylalanine, m-19F-phenylalanine, m-19F-tyrosine, and 19F-methylhistidine. Incorporation of difluoromethionine,34 hexafluoroleucine,35a (2S,4R)-5-19F-leucine,35b and 19F-proline35c has been reported. These other analogs will provide a valuable tool for the investigation of protein structure and folding. The recent discovery of a fluorinase36 may allow other fluorine-labeled amino acids to be synthesized enzymatically. Currently only 19F-phenylalanine can be incorporated site specifically as described above. On the other hand, Wang et al.37 reported a method for site-specific incorporation of O-methyl tyrosine suggesting that it may be possible to incorporate 19F-tyrosine site specifically. The chapter by Gerig5 includes a number of suggestions for the incorporation of fluorine labels into proteins. The reader would be well served by consulting this reference. Site-Specific Labeling with Cysteine-Reactive Compounds
Another method that has been used for site specifically labeling proteins with fluorine for 19F-NMR experiments is the use of compounds that are reactive to cysteine residues. This approach was pioneered by Gerig5 and has the advantage that one can probe a single site. Obviously, the effect of the cysteine mutation, before and after treatment with a fluorinated compound, must be taken into account. However, a major advantage of cysteine labeling is that one can use these sites to probe with other cysteine-reactive reagents, such as fluorescent or spin labels, for comparative purposes. A recent study of the integral membrane protein rhodopsin has shown that fluorine labeling of two cysteine residues that are close in space can allow one to obtain through-space information under different conditions (i.e., light and dark) using 19F nuclear Overhauser effects (NOEs).38 34
M. D. Vaughn, P. Cleve, V. Robinson, H. S. Duewel, and J. F. Honek, J. Am. Chem. Soc. 121, 8475 (1999). 35 (a) Y. Tang and D. A. Tirrell, J. Am. Chem. Soc. 123, 11089 (2001). (b) J. Feeney et al., J. Am. Chem. Soc. 118, 8700 (1996). (c) C. Renner et al., Angew Chem. Int. Ed. 40, 923 (2001). 36 D. O’Hagan, C. Schaffrath, S. L. Cobb, J. T. Hamilton, and C. D. Murphy, Nature 416, 279 (2002). 37 L. Wang, A. Brock, B. Herberich, and P. G. Schultz, Science 292, 498 (2001). 38 M. C. Loewen et al., Proc. Natl. Acad. Sci. USA 98, 4888 (2001).
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TABLE II Cysteine-Reactive Fluorinating Reagents Formula
Name; Commercial availability
Reference
CF3-CH2-SH CF3-CO-CH2-Br F-C4H4-SH CF3-C4H4-NH-CO-CH2l (CF3)3C-C4H4-NH-CO-CH2l
2,2,2-Trifluoroethanethiol; yes 3-Bromo-1,1,1-trifluoroacetone; yes 4-Fluorobenzenethiol; yes 4-(Trifluoromethyl)phenyliodoacetamide; no 4-(Perfluoro-tert-(butyl)phenyliodoacetamide; no
38, 39 41, 42 43 44 45
There are a number of different compounds that can be used to label cysteines, and some recently used ones are presented in Table II. Although several cysteine-reactive fluorocompounds have been used recently for 19 F-NMR studies, a highly selective reagent for cysteine is the use of trifluoroethylthiol (TET).38,39 Whereas a fluorohalogenated compound may in some cases show reactivity toward amines, TET labeling of cysteine is specific for the formation of a disulfide. TET labeling produces an analog that is similar in length to lysine and chemically similar to methionine, such that either substitution [Lys to Cys (TET) or Met to Cys (TET)] would probably result in minimal perturbation of the molecule. Also, this compound exhibits a sharp resonance, due to the lack of proton coupling of the CF3 group.40–45 Conclusions
This chapter outlines the methods that can be used to incorporate fluorine-labeled amino acids, as well as other ligands, into proteins. There are many reasons for using these techniques to study the structural and dynamic aspects of proteins. Of these the most dramatic is the sensitivity of the fluorine nucleus to its environment under conditions in which there are only minimal perturbations in the wild-type structure. Thus, studies can investigate the changes due to ligand binding, protein–protein interactions, as well as protein folding. Currently no good theory exists for 39
J. Klein-Seetharaman, E. V. Getmanova, M. C. Loewen, P. J. Reeves, and H. G. Khorana, Proc. Natl. Acad. Sci. USA 96, 13744 (1999). 40 G. R. Drapeau, W. J. Brammar, and C. Yanofsky, J. Mol. Biol. 35, 357 (1968). 41 M. R. Thomas and S. G. Boxer, Biochemistry 40, 8588 (2001). 42 K. Oxenoid, F. D. Sonnichsen, and C. R. Sanders, Biochemistry 41, 12876 (2002). 43 J. P. Caradonna, E. W. Harlan, and R. H. Holm, J. Am. Chem. Soc. 108, 7856 (1986). 44 J. W. Shriver and B. D. Sykes, Biochemistry 21, 3022 (1982). 45 D. Heintz, H. Kany, and H. R. Kalbitzer, Biochemistry 35, 12686 (1996).
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relating chemical shifts to the structural environment. When such a theory is developed, the amount of information that can be obtained from fluorine chemical shifts should be impressive. Appendix I: General Method for the Production of Proteins Site Specifically Labeled with p-19F-Phenylalanine Using PapD as an Example
Originally, the procedure described by Furter for labeling mouse dihydrofolate reductase was to grow the bacteria in M9 minimal media supplemented with phenylalanine/p-19F-phenylalanine (0.2 and 3 mM, respectively).31 This serves the purpose of keeping a selective pressure to maintain the resistance to p-19F–labeled phenyalanine. In the original paper,31 the cells were grown to an A600 of 1.0 and then shifted to media containing 3 mM p-19F-phenylalanine and 0.03 or 0.04 mM phenylalanine. It was found that the level of specific p-19F-phenylalanine incorporation increased with ‘‘an unproportional 2.3-fold increase in p-19F-phenylalanine contamination’’ when using 0.03 versus 0.04 mM phenylalanine. Because the level of specific incorporation is important, we use the 0.04 mM phenylalanine final concentration for labeling, and find very similar levels of specific incorporation over uniformly labeled protein. A modified protocol for site-specific labeling is described below and should be applicable to other proteins. Two days prior to the growth of bacteria, frozen K10F6 cells harboring the pQE80papDPhe!Amber mutant/pRO117 are streaked onto LB agar plates containing 100 g/ml ampicillin and 50 g/ml kanamycin. A single colony is picked and grown the morning of the following day in 5 ml of LB containing antibiotics at 37 . In the evening, the cells are diluted 1:1000 into 100 ml of the defined media containing 0.2 mM Phe/3 mM p-19F-phenylalanine with antibiotics. The defined media we use30 contains (for a 1-liter volume) 0.2 mM phenylalanine/3 mM p-19F-phenylalanine, 0.5 g alanine, 0.4 g arginine, 0.4 g aspartate, 0.05 g cystine, 0.4 g glutamine, 0.65 g glutamate, 0.55 g glycine, 0.1 g histidine, 0.23 g isoleucine, 0.23 g leucine, 0.42 g lysine hydrochloride, 0.25 g methionine, 0.1 g proline, 2.1 g serine, 0.23 g threonine, 0.17 g tyrosine, 0.23 g valine, 0.5 g adenine, 0.65 g guanosine, 0.2 g thymine, 0.5 g uracil, 0.2 g cytosine, 1.5 g sodium acetate, 1.5 g succinic acid, 0.5 g NH4Cl, 0.85 g NaOH, and 10.5 g K2PO4 per 950 ml of H2O. This is then autoclaved. To this is added 50 ml of a sterile-filtered solution of 40% glucose, 4 ml of 1 M MgSO4, and 10 ml of a sterile solution of 2.7 mg FeCl36H2O, 2 mg CaCl22H2O, 2 mg ZnSO47H2O, 2 mg MnSO4H2O, 50 mg l-tryptophan, 50 mg thiamine, 50 mg niacin, and 1 mg biotin. This solution has a pH
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near 7.2. The antibiotics (1 ml of 50 mg/ml kanamycin, 1 ml of 100 mg/ml ampicillin) are added separately. We have found that it is important to maintain the bacteria in a logarithmic phase of growth and not to let them go into stationary phase, as we have observed premature lysis of the bacteria. Thus, the overnight culture is started rather late (7:00 pm). The bacteria from the overnight culture are then diluted 1:50 into 750 ml of media in Fernbach flasks. The cells are grown to an A600 of 1.0, and harvested by centrifugation. Once harvested, the cells are then washed twice with 0.9% NaCl, 3 mM p-19F-phenylalanine. The resuspended cells are grown for about 20–30 min. min. Then, IPTG is added to a final concentration of 1.0 mM, and the cells are grown for an additional 1–3 h. Following this, the usual procedures for purifying the protein of choice are applicable.