19F NMR studies of solvent exposure and peptide binding to an SH3 domain

Biochimica et Biophysica Acta 1770 (2007) 221 – 230 www.elsevier.com/locate/bbagen

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F NMR studies of solvent exposure and peptide binding to an SH3 domain Ferenc Evanics a , Julianne L. Kitevski a , Irina Bezsonova b,c , Julie Forman-Kay c,d , R. Scott Prosser a,⁎ a

Department of Chemistry, University of Toronto, UTM, 3359 Mississauga Rd. North Mississauga, ON, Canada L5L 1C6 b Department of Chemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 c Molecular Structure and Function, Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8 d Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8 Received 28 June 2006; received in revised form 2 October 2006; accepted 23 October 2006 Available online 11 November 2006

Abstract 19

F NMR was used to study topological features of the SH3 domain of Fyn tyrosine kinase for both the free protein and a complex formed with a binding peptide. Metafluorinated tyrosine was biosynthetically incorporated into each of 5 residues of the G48M mutant of the SH3 domain (i.e. residues 8, 10, 49 and 54 in addition to a single residue in the linker region to the C-terminal polyhistidine tag). Distinct 19F NMR resonances were observed and subsequently assigned after separately introducing single phenylalanine mutations. 19F NMR chemical shifts were dependent on protein concentration above 0.6 mM, suggestive of dimerization via the binding site in the vicinity of the tyrosine side chains. 19F NMR spectra of Fyn SH3 were also obtained as a function of concentration of a small peptide (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Prodiaminopropionic acid –NH2, known to interact with the canonical polyproline II (PPII) helix binding site of the SH3 domain. Based on the 19F chemical shifts of Tyr8, Tyr49, and Tyr54, as a function of peptide concentration, an equilibrium dissociation constant of 18 ± 4 μM was obtained. Analysis of the line widths suggested an average exchange rate, kex, associated with the peptide–protein two-site exchange, of 5200 ± 600 s− 1 at a peptide concentration where 96% of the FynSH3 protein was assumed to be bound. The extent of solvent exposure of the fluorine labels was studied by a combination of solvent isotope shifts and paramagnetic effects from dissolved oxygen. Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the Tyr on the C-terminal tag, appear to be fully exposed to the solvent at the metafluoro position in the absence of binding peptide. Tyr54 and, to some extent, Tyr10 become protected from the solvent in the peptide bound state, consistent with known structural data on SH3–domain peptide complexes. These results show the potential utility of 19F-metafluorotyrosine to probe protein–protein interactions in conjunction with paramagnetic contrast agents. © 2006 Elsevier B.V. All rights reserved. Keywords: Fluorine NMR; Fyn SH3; Solvent exposure; Oxygen; Paramagnetic effect

1. Introduction The spectroscopic study of protein–peptide interactions ideally should utilize probes that are non-perturbing but accurately reflect both the equilibrium and kinetic features of Abbreviations: Gd(III)DTPA-BMA, Gd(III)-diethylenetriamine pentaacetic acid-bismethylamide; NiEDDA, nickel (II) ethylenediaminediacetate; NOESY, Nuclear Overhauser Effect SpectroscopY; PPII, polyproline type II; ROESY, Rotating frame Overhause Effect SpectroscopY; CW, continuous wave; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl ⁎ Corresponding author. Tel.: +1 905 828 3802; fax: +1 905 828 5425. E-mail address: [email protected] (R.S. Prosser). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.10.017

the interaction. Fluorine NMR is an excellent technique for the study of such phenomena since the isotopic fluorine label is generally weakly perturbing while the spectra are sensitive to changes in van der Waals or electrostatic environments expected with binding. Furthermore, the resonances of the bound and free states are often significantly different, providing an excellent dynamic range for the study of equilibria and kinetics. 19F NMR is also amenable to approaches involving the addition of paramagnetic contrast agents for purposes of studying solvent exposure and possible protection from solvent exposure due to binding interactions. In this paper we explore the use of metafluorotyrosine as an NMR probe of an interaction between

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the SH3 domain of Fyn tyrosine kinase and a proline-rich peptide. Issues of 19F labeling and protein expression are considered, while strategies for assignment and interpretation of data in terms of dynamics, protein dimerization, protein– peptide interactions, and solvent exposure are provided. 1.1. Solvent exposure In protein structure studies, the determination of solvent exposure by spectroscopic methods is a longstanding problem. For example, fluorescence or ESR experiments monitor solvent exposure by the addition of appropriate water soluble additives which serve to quench or broaden signals in proportion to the collisional frequency of the quencher [1–5]. In NMR, solvent exposure has been measured by monitoring signal from exchangeable groups upon substituting water with D2O [6,7], solvent NOESY or ROESY schemes [8–11], or relaxation experiments in the presence of soluble paramagnetic additives such as Gd(III)DTPA-BMA [12] and TEMPOL [13,14]. In general for any spectroscopic method the measurement of solvent exposure is complicated by the hydration shell and a vast range of exchange timescales of water with various residues. Moreover, in situations where relaxation agents are used, the effect may explicitly depend on the geometry and diffusion rate of the paramagnet and potential preferential interactions with specific residues, local surface hydrophobicity, and local dynamics. Solvent induced isotope shifts or changes in homonuclear and heteronuclear scalar coupling constants resulting from the exchange of H2O with D2O have also been explored in studies of solvent exposure in proteins. Although such effects are difficult to observe by 13C, 1H, or 15N NMR [15,16], solvent induced isotope shifts may be as large as 0.25 ppm in 19F NMR spectra, offering a unique way to probe solvent exposure [17– 20]. Such experiments may be corroborated by T1 measurements of the 19F probe, where it is commonly observed that a high spin-lattice relaxation rate in D2O is indicative of burial in the hydrophobic protein interior which serves as a significant source of dipolar relaxation. Intermolecular 1H–19F NOEs are also efficient indicators of solvent exposure, particularly in cases where H2O is strongly bound [21]. Although 19F NMR chemical shifts are extremely difficult to predict based on environment [22,23], soluble paramagnets are useful in 19F NMR studies of solvent exposure. One such paramagnet which exhibits pronounced relaxation enhancement and chemical shift perturbations is molecular oxygen [24,25]. In a recent study of a membrane protein in detergent micelles, oxygen-induced chemical shift perturbations from specifically fluorinated sites of a transmembrane (TM) domain belonging to a polytopic TM protein provided information on secondary structure, helix– helix interfaces, and immersion depth [26]. Despite the sensitivity arising from the use of dissolved oxygen as a contrast agent, preferential interactions and local dynamics of both the additive and protein complicate analysis of effects in terms of solvent exposed surface areas [27]. Ideally, solvent exposure might be better assessed by separately measuring the effects of a hydrophilic and hydrophobic contrast

agent, whose sizes and mobilities are comparable. Here we define a contrast agent as any additive (paramagnetic or otherwise) which affects a change in chemical shift or relaxation properties of labels of interest in a spatially dependent manner. Thus, in the context of 19F NMR, D2O can be thought of as a contrast agent since its substitution for H2O causes local peak shifts proportional to solvent exposure. ESR studies have used complementary paramagnetic contrast agents such as O2 and Ni (II) to separate steric effects and partitioning effects in proteins [5,28]. We extend this idea to 19F NMR which has the distinct advantage that it is sensitive to two simple contrast agents, oxygen and H2O/D2O, whose sizes are similar while oxygen is hydrophobic and water is polar. Effects from oxygen are easily measured through chemical shift perturbations and spin-lattice relaxation rates, while contrast effects from water may be measured through 1H–19F NOEs or solvent induced isotope shifts, resulting from exchanging H2O with D2O. 1.2. Fyn tyrosine kinase and SH3 domains Fyn tyrosine kinase plays a key role in signal transduction processes. The protein's catalytic domain is adjacent to two small domains (the so-called Src-homology regions 2 and 3 or SH2 and SH3 domains) which modulate signal transduction events in the cell through binding interactions [29,30]. The 59 residue SH3 domain is present in many eukaryotic proteins which are involved in signal transduction, cell polarization and membrane–cytoskeleton interactions. The SH3 domain often functions as an intramolecular regulator of kinase activity, while serving as a targeting moiety to direct Src kinases to the proper intracellular sites. The Fyn SH3 domain shows the typical SH3 topology consisting of five β-strands as shown in Fig. 1. A binding surface surrounded by the n-Src and RT-loops consists predominantly of aromatic residues which are known to interact with proline-rich targets [31,32]. Tyrosines figure prominently in this binding surface, including tyrosines 8, 10, and 54 in Fyn, which are highly conserved among SH3 domains [33]. The SH3

Fig. 1. Ribbon diagram of the Fyn SH3 domain showing the location of the four metafluorotyrosines which sit in the peptide binding groove.

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domain possesses a fourth tyrosine residue at position 49, somewhat removed from the binding pocket. Proteins or peptides interacting with the Fyn SH3 domain usually exhibit dissociation constants in the range of 1–10 micromolar for binding consistent with transient interactions, as might be expected for regulation of signaling. However, synthetic peptides have been designed to bind to the Fyn SH3 domain with nanomolar dissociation constants [34]. This paper explores the topology of the SH3 domain of Fyn and subsequent changes upon peptide binding using 19F NMR. We make use of a well studied destabilized mutant version of the Fyn SH3 domain (i.e. G48M) which exists in fast equilibrium with an unfolded state via a well-defined intermediate under native conditions at room temperature [35,36]. However, the work discussed in this paper was performed under low temperature conditions (10 °C) where the unfolded state is very weakly populated (< 1%) and the folded state dominates. Via fluorinated tyrosine probes, local side chain mobility, inter-residue contacts through NOEs, solvent exposure, and the ensuing changes upon addition of the binding peptide are considered. Details of the interaction between the peptide and the protein are reliably obtained in terms of the binding equilibrium constant and effective exchange rate by monitoring the behavior of the chemical shifts and line widths, as a function of peptide concentration. 2. Theory 2.1. Solvent exposure

dynamics after titrating a target peptide to the Fyn SH3 domain. The analysis of the ensuing chemical shift and line width changes, in terms of binding and dynamics, is discussed below. 2.2. Protein–peptide interactions 19

F NMR is ideally suited to the study of peptide binding in part because very large chemical shift perturbations and relaxation rate changes are often observed upon binding. Furthermore, a global fit of all chemical shift titration curves and relaxation rate analyses from multiple sites in the binding pocket should result in a single dissociation constant and exchange time. Assuming that the resonances associated with both the free protein and fully saturated bound protein are distinct, we may describe the dependence of the chemical shift, δ, on peptide concentration, [p], by, d¼

A  ½p ; ½p þ Kd

ð1Þ

where A is a fitting constant and Kd defines the equilibrium dissociation constant. In the event of intermediate timescale exchange, line broadening can be slightly more complicated. However, if we assume for the moment that the bound state is much more populated over the concentration range considered, the transverse relaxation rate associated with the bound peak may be given as [37–39] P0

In describing the chemical shift perturbation or relaxation rate enhancement arising from a diffusible paramagnet such as dissolved oxygen, the key variables include the collisionally accessible exposed surface area and the local concentration of the contrast agent, which has been shown to depend on surface hydrophobicities [27]. Ideally, the paramagnetic shift, ΔδO2, or relaxation rate, R1P, should be normalized by dividing the observed effect (shift or rate) by the equivalent result for a fully exposed probe. For example, Fyn SH3 possesses a tyrosine residue on the C-terminal polyhistidine tag, which is expected to exhibit the largest degree of solvent exposure. Thus R1P or ΔδO2 acting on Tyr8, 10, 49, or 54 of Fyn SH3 can be normalized by dividing the result by that observed for the Tyr probe on the exposed tag. We may similarly define the normalized version of the chemical shift perturbation due to the solvent isotope effect. Though this is not of paramagnetic origin, we nevertheless expect the normalized solvent isotope shift to depend on local solvent accessibility and local hydrophobicity. A ratio of the normalized paramagnetic effect from oxygen to the equivalent normalized solvent isotope effect should then reflect the local hydrophobicity, assuming accessibilities of water and oxygen are similar. Herein, we measure the 19 F paramagnetic shifts and relaxation rate enhancement arising from dissolved oxygen in addition to solvent isotope shifts of Fyn SH3 enriched with metafluorinated tyrosine, in order to better address solvent exposure and hydrophobicity of the tyrosine side chains. We then consider the changes in solvent exposure and side chain

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R21 ¼ R2 þ

kex 2

1 2 2 2 1=2 1=2 Dx2 þ½ðkex þDx2 Þ2 16p1 p2 Dx2 kex  g ;  pffiffiffi fkex 8 ð2Þ where we envisage a two-site exchange between a bound state, 1, and a free protein state, 2. Δω represents the difference in radial frequency units between the fully bound and fully free P0 resonances, R2 represents the population averaged relaxation rate in the absence of exchange, and the overall sum of exchange rates, kex, between sites 1 and 2 is given by kex ¼ k12 þ k21 :

ð3Þ

The relative fraction of the complexed and free states of the Fyn SH3 domain may further be expressed in terms of the forward and reverse rate constants as p1 = k21 / kex and p2 = k12 / kex. Thus the strategy in determining the equilibrium dissociation constant is to monitor the chemical shifts of resonances of the tyrosine residues involved in binding as a function of peptide concentration. The effective exchange rate, kex, may be similarly analyzed as a function of peptide concentration. 3. Materials and methods 3.1. Sample preparation A plasmid coding for the isolated G48M mutant of the isolated Fyn SH3 domain, residues 85–142, cloned in such a way that 5 additional residues

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(MVQIS) were added to the N-terminus and 16 (RLDYKDDDDKHHHHHH) added to the C-terminus, was transfected into E. coli strain BL21(DE3) under the control of the T7 promoter. Expression of the 15N labeled protein was induced for 3 h at an OD600 of 0.8 by addition of 250 mg/L IPTG to bacterial growths at 37 °C in M9 minimal medium, supplemented with 0.3% D-glucose, 0.1% 15NH4Cl, 100 mg/L ampicillin, 10 mg/L thiamine, 10 mg/L biotin, 1 mM MgSO4 and 1 mM CaCl2. Uniform fluorotyrosine labeling was achieved by introducing glyphosate (1 g/L), phenylalanine (50 mg/L), tryptophan (50 mg/L), and 19F labeled metafluoro (L, D) tyrosine (70 mg/L) (Sigma Chemicals Mississauga, ON) to the bacterial culture 1 h before induction (OD600 ∼ 0.4). Unlike prior methods [40], no auxotroph was found to be necessary to obtain reasonable yields (24 mg/L). Cells were harvested 3 h after induction by centrifugation, and lysed by sonication in 0.1 M NaH2PO4, 0.01 M Tris–HCl, 6 M guanidine hydrochloride (GdmCl), and 20 mM imidazole. The Fyn SH3 domain was purified on a nickel ion exchange column at 25 °C under denaturing conditions (6 M GdmCl) then refolded by dialysis against 10 mM Tris–HCl, 0.2 mM EDTA, and 250 mM KCl. For NMR measurements, the protein concentration was stabilized in 20 mM Tris–HCl buffer. Four mutants in which phenylalanine was substituted for tyrosine at position 8, 10, 49 or 54 were made using a Quick Change Kit (Stratagene, CA, USA). Protein expression, purification, and NMR sample preparation of the mutants was performed as described above. Protein concentrations were determined by absorbance measurements, using a known extinction coefficient of 60.5 μM− 1 cm− 1. The concentration of the “wild type” G48M Fyn SH3 domain sample, which was used for the bulk of the solvent exposure and peptide binding studies, was estimated to be 0.4 mM which was found to be below the threshold for oligomerization. The phenylalanine mutant concentrations ranged from 0.61 to 0.69 mM.

3.2. NMR experiments A 400 μL sample volume was deemed sufficient for shimming purposes, using a 5 mm OD, 3 mm ID sapphire NMR sample tube (Saint Gobain-Saphikon Crystals, Milford, NH, USA), designed to tolerate pressures as high as 270 bar. To measure effects of dissolved oxygen, the sample was first equilibrated at 10 °C outside the magnet at an oxygen partial pressure of 20 bar for 2 days, then equilibrated overnight in the magnet at the desired partial pressure of 20 bar. Using open Swagelok (Swagelok, Solon, OH, USA) connections to a pressurized oxygen supply, it was possible to maintain the pressure during the entire course of the NMR experiment. To reliably reproduce oxygen concentrations, we relied on the measurement of the 1H T1 of water which was typically 100 ms at 20 bar (PO2). Upon completing oxygen experiments, the sample was degassed by first transferring it to a 1.5 mL microfuge tube resting on an ice bath, after which it was slowly stirred using a sterile needle tip which precipitated the bubbling of oxygen. The sample was then left for 24 h in a nitrogen environment to allow for residual degassing. To exchange H2O for 2 H2O buffer, a 0.5 mL centrifugal concentrator with a molecular weight cutoff of 3 kDa was used. A significant amount of protein (30–40%) was lost upon solvent exchange and transfer back to the original sapphire NMR tube, resulting in a need for greater signal averaging in the 2H2O sample. Finally, 0.008 mg of 4hydroxy-TEMPO (Sigma Chemicals, Mississauga, ON), also known as TEMPOL, was added to the sample to measure contrast effects through T1 from a dissolved paramagnet. 1H,15N gradient selected HSQC and 19F onedimensional NMR experiments were performed at 10 °C on a 600 MHz Varian Inova spectrometer, using a standard 5 mm HCN triple resonance single gradient solution NMR probe, in which the high frequency channel could be tuned to either 19F or 1H. 8 scans and 80 increments spanning 1650 Hz in the indirect dimension were used to obtain the HSQC spectrum while 512 scans were typically used to obtain the 19F NMR spectrum. The measurement of 19F spinlattice relaxation times was accomplished by an inversion recovery sequence (i.e. 180 – τ – 90) using a total of 8 τ values, logarithmically spaced between 10 ms and 5 s (ambient sample) or 1 ms and 1 s (oxygenated sample). The repetition time was adjusted to either 6.5 s (ambient sample) or 1.5 s (oxygenated sample). Two Hahn echo refocusing pulses, spaced by 350 μs, were appended to all 19F NMR sequences, to help remove background 19F signal from the probe. One-dimensional homonuclear 19F NOESY experiments were performed using mixing times of 100 ms, 250 ms, 500 ms, and 1000 ms in order to measure interactions between the tyrosine labels in the protein. In these

experiments, each distinct 19F resonance (in addition to a dummy frequency several hundred Hz from the nearest resonance) was separately saturated via an appropriate low power CW pulse during the mixing time and the subsequent signal was then compared to an equivalent off-resonance saturation. NOE intensities were approximately 20% the total intensity of the individual unsaturated resonances at a mixing time of 500 or 1000 ms and roughly 10% the total intensity at a mixing time of 100 ms. A two-dimensional homonuclear 19F NOESY experiment was also performed using a mixing time of 250 ms (data not shown). An 15N-edited two-dimensional (1H,1H) NOESY was also performed to help assign HSQC peaks in the fluoro-tyrosine substituted protein.

4. Results and discussion 4.1. Effect of introducing metafluorotyrosine at residues 8, 10, 49, and 54 Fig. 2 compares the (1H,15N) HSQC NMR spectrum of 15N enriched Fyn SH3 (blue contours) with that of the fluorotyrosine substituted version (red contours). Note that there is no detectable 15 NH tyrosine signal from the fluorotyrosine substituted species, suggesting that 19F-tyrosine incorporation was at least 95%, based on the signal to noise ratio in the HSQC spectrum. Furthermore, incomplete biosynthetic labeling would be expected to give rise to a multiplicity of 19F NMR peaks since the tyrosine residues are quite close to each other in the Fyn SH3 domain. The two spectra of the 15N-enriched fluorinated and 15N-enriched Fyn SH3 domains, which were obtained at 0.4 mM concentrations, are sufficiently similar to conclude that the overall folds are the same. However, there are

Fig. 2. (1H,15N) HSQC NMR spectrum of 15N enriched Fyn SH3 domain (shown in blue) overlayed with the equivalent spectrum of the Fyn SH3 domain in which all five tyrosines were replaced with metafluorotyrosine (shown in red). Both spectra were acquired at 10 °C. The circled peaks indicate the resonances associated with each of the 4 tyrosine resonances, which can only be seen in the control spectrum (i.e. fluorotyrosine-free). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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modest differences in chemical shifts for a significant number of residues. Fluorination in aromatic amino acids is known to result in a redistribution of partial charge across the ring, a decreased hydrogen bond capacity (of the fluorine atom) and a slight increase in the van der Waals radius in the vicinity of the CF group [41] while more significant effects may occur in the case of perfluorination of aromatic groups [42]. Such changes may give rise to slight structural perturbations and possibly large functional perturbations in the case of enzymes where tyrosine directly participates in the in the reaction mechanism [17,43–45]. 4.2. Assignment of

19

F resonances and side-chain interactions

Fig. 3 presents the 19F NMR spectrum of the fluorotyrosine substituted Fyn SH3 domain for the “wild-type” G48M and the four Tyr-to-Phe mutants used for the assignment. All spectra were acquired at 10 °C under similar conditions and at a protein concentration of approximately 0.6 mM, with the exception of the top most spectrum which was obtained from a 0.4 mM sample of the fluorotyrosine substituted Fyn SH3 domain, and which contained a small amount of free metafluorinated tyrosine intended as a chemical shift reference. All five resonances associated with fluorotyrosine sites on the G48M mutant of the Fyn SH3 domain (i.e. Tyr8, Tyr10, Tyr49, Tyr54, and the tyrosine on the C-terminal tag) can be clearly identified. Note that the sharp central peak at − 137.32 ppm arises from the fluorotyrosine label on the C-terminal tag. This label potentially serves as a useful internal chemical shift and relaxation rate reference, both for the study of solvent exposure via solvent isotope shifts or paramagnetic rates arising from dissolved oxygen or other paramagnetic additives (vide infra). The

Fig. 3. 19F NMR spectra of the metafluorotyrosine substituted Fyn SH3 domain at 10 °C. The top spectrum represents the fully substituted species at a concentration of 0.4 mM and consisting of identical 19F isotopic labels at Tyr8, Tyr10, Tyr49, Tyr54, and the tyrosine in the C-terminal tag. A small amount of free metafluorotyrosine was added as an internal control for the solvent exposure studies. The spectrum labeled “conc” represents a more concentrated version of the Fyn SH3 domain (0.6 mM) where the dimeric state is believed to dominate. Additional 19F NMR spectra in the figure obtained under identical conditions, represent single phenylalanine mutants, as labeled, whose concentrations ranged between 0.6 mM and 0.7 mM.

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spectrum of the Fyn SH3 domain obtained under dilute conditions exhibits the greatest chemical shift dispersion and this is particularly true of the Tyr54 and Tyr10 resonances which appear to exhibit a shift variation of approximately 0.5 ppm depending on protein concentration. For example, the upfield resonances associated with Tyr10 in Fig. 3 appear to adopt a shift of either − 138.25 ppm or − 138.75 ppm and, in most cases, there appears to be a coexistence of the two resonances which depends on concentration. Similarly, Tyr54 exhibits chemical shifts between − 137.25 ppm when concentrated and − 136.75 ppm under more dilute conditions. This dramatic concentration dependence of chemical shifts is suggestive of the formation of a dimer of the G48M mutant of FynSH3, mediated by the fluorotyrosine sidechains. Single tyrosine to phenylalanine mutations (Y54F, Y49F, Y10F, and Y8F) appear to alter the equilibrium between dimer and monomer which we tentatively associate with the spectrum of the Fyn SH3 domain at 0.4 mM or less (top most spectrum of Fig. 3). Selective saturation of the major peak of Tyr10 in the top most spectrum of Fig. 3 results in the gradual decrease in the minor peak. Based on the steady state value of the minor peak and accounting for the (identical) T1 of both peaks, we estimate the forward rate constant associated with the formation of the dimer to be 1.16 s− 1 [46]. However, the majority of the mutants (Y8F, Y10F, and Y49F) give rise to a spectrum which we attribute to a dimeric state of FynSH3 at the concentrations used in these experiments (i.e. 0.6 mM or higher). There appears to be a network of interactions between the tyrosine side chains, which are perturbed by the introduction of even a single phenylalanine residue. The origin of these side-chain interactions is not exclusively intramolecular, as evidenced by the significant concentration dependence associated with the chemical shifts of Tyr10 and Tyr54. No such dimerization or clustering is known to occur with non-fluorinated versions of the Fyn SH3 domain or the G48M mutant of the FynSH3 domain, suggesting that the fluorinated side chains are responsible for the dimerization of the Fyn SH3 domains, at least in the absence of a binding peptide. Clusters of fluorinated residues are well known to exhibit a so-called fluorophobic effect which is stronger than hydrophobic forces alone in directing association of fluorinated interfaces [47–50]. This property has been applied previously in de novo protein design, where biosynthetically fluorinated species introduced to the hydrophobic core have proven to render the protein more resistant to heat denaturation [49,50]. In the case of the Fyn SH3 domain, all 5 fluorotyrosines are located on the protein exterior and at least three of these residues reside along a single surface (Fig. 1). Therefore, at concentrations of 0.6 mM or higher, the protein is believed to form a transient dimer as evidenced by the significant concentration dependent chemical shift perturbation associated with Tyr 10 and Tyr 54. Though a thorough series of spectra as a function of concentration was not obtained, we estimate that 0.4 mM represents the threshold concentration above which a dimeric state is obtained. The lack of any significant changes in the spectrum above 0.6 mM suggests that higher order oligomerization states are not obtained, which is consistent with the notion that the dimerization interface exists

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from a relayed NOE effect or from greater mobility of the Tyr10 side chain. Note that the figure is meant to serve as a guide of approximate positioning of the tyrosine residues since a slight variation in the side chain torsion angles can significantly alter the relative separation of the fluorine nuclei. Surprisingly, there are significant NOE contacts between all of the residues and the fluorotyrosine residue on the C-terminal tag. A significant fraction of these NOE contacts may arise from intermolecular effects, established through dimerization, and a thorough analysis of cross peak intensity as a function of mixing time and concentration should provide an estimate of the equilibrium constant and exchange time. 4.3. Solvent exposure of the fluorotyrosine residues for the free Fyn SH3 domain

Fig. 4. 19F homonuclear NOESY amplitudes obtained for a mixing time of 100 ms, upon selective saturation of each resonance. Negative NOEs are indicated by a solid line while positive NOEs are indicated by a dashed line. The line thickness corresponds to the relative magnitude of the NOE, while the source of each arrow indicates the residue which was saturated. Note that the tyrosine in the C-terminal tag is designated as C-term in this figure.

on the polyproline II interacting interface and is mediated by the fluorotyrosine side chains. Evidence that Tyr54 is necessary for dimerization is also found in the spectrum of the Y54F mutant, shown in Fig. 3, which we have assigned as a monomeric state, as evidenced by the chemical shift of Tyr10. An extensive network of inter-side chain interactions is also seen through 19F,19F NOESY measurements, where the NOE intensities are given in Fig. 4. All NOEs were obtained by separately irradiating each of the above resonances for 100 ms and observing the change in peak intensity with respect to an off resonance irradiation. Here, the magnitude of the NOE is indicated by the thickness of the line, while solid and dashed lines indicate negative and positive NOEs, respectively. Residues 8, 54, and 49, which seem to lie along a groove in the Fyn SH3 domain, all appear to establish prominent NOE contacts, while the positive NOEs to residue 10 may arise either

Table 1 presents the results of several 19F NMR experiments designed to address the extent of solvent exposure for the fluorotyrosine side chains of the Fyn SH3 domain. Note that in D2O, where dipolar relaxation arises only from the protein, 19F spin-lattice relaxation times associated with residues 8, 10, 49, and 54 and the C-terminal tag range between 0.9 s and 1.1 s. A small R1 in D2O is indicative of fewer protein contacts and, thus, greater solvent exposure [17]. It is not surprising that all tyrosine relaxation rates fall in such a narrow range in the absence of binding peptide, since none are buried in the hydrophobic core of the Fyn SH3 domain. Fig. 5A shows a series of 19 F NMR spectra of the metafluorotyrosine enriched Fyn SH3 domain as a function of H2O/D2O, where the corresponding shifts are given in Table 1. To enable assignments to be followed, the 19F NMR spectrum was obtained in mostly H2O (10% D2O), 50% D2O, and 100% D2O. The solvent isotope shift could then be determined from the difference of the chemical shifts associated with the 50% D2O and 10% D2O sample (i.e. ΔδHDO-H2O) and between 100% D2O and the 10% D2O mixture (i.e. ΔδD2O-H2O). Ideally the isotope shift, ΔδD2O-H2O, should be proportional to ΔδHDO-H2O, unless differences in conformation arise for high D2O fractions. As can be seen from Table 1, the two shifts (ΔδD2O-H2O and ΔδHDO-H2O) are proportional to each other within a 25% margin with the exception of the solvent isotope shift associated with

Table 1 Chemical shifts, line widths, and relaxation rates and associated changes upon substitution of H2O with D2O or addition of dissolved oxygen for all fluorotyrosine resonances in both the free protein and protein saturated with binding peptide δ[ppm]

Δν1/2 [Hz]

R1 [s− 1]

ΔδDHO-H2O [ppm]

ΔδD2O-H2O [ppm]

ΔδD2O-H2O [ppm]

R1, D2O [s− 1]

ΔδO2 [ppm]

RP1 [s− 1]

− 136.686 − 137.093 − 137.372 − 137.665 − 138.717 − 134.179 − 136.825 − 137.408 − 136.365 − 138.737

51.640 43.360 47.750 61.520 57.660 116.857 54.542 44.934 98.285 71.641

1.4 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 1.3 ± 0.1 2.0 ± 0.1 2.1 ± 0.1 1.3 ± 0.1 1.8 ± 0.1 1.6 ± 0.1 1.9 ± 0.1

− 0.225 − 0.081 − 0.113 − 0.108 0.071 N/A N/A N/A N/A N/A

− 0.113 − 0.113 − 0.100 − 0.094 − 0.021 N/A N/A N/A N/A N/A

−0.338 −0.194 −0.214 −0.202 0.050 N/A N/A N/A N/A N/A

1.1 ± 0.1 1.1 ± 0.1 1.1 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 N/A N/A N/A N/A N/A

−0.139 −0.119 −0.082 −0.107 −0.069 −0.085 −0.115 −0.089 −0.101 −0.056

5.6 ± 0.1 3.6 ± 0.1 2.7 ± 0.1 3.6 ± 0.1 3.6 ± 0.1 0.3 ± 0.1 4.4 ± 0.1 2.1 ± 0.1 3.0 ± 0.1 1.6 ± 0.1

Note that ΔδDHO-H2O represents the solvent induced isotope shifts upon substituting 90/10 H2O/D2O with 50/50 H2O/D2O while ΔδD2O-DHO represents the solvent induced isotope shifts upon substituting 50/50 H2O/D2O with 100% D2O.

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ΔδO2, and relaxation enhancements, (R1P≡R1,O2 − R1), are given in Table 1. Within a 20% margin, both measures of local oxygen content consistently reveal that meta positions of the fluorotyrosine side chains are fully exposed to the solvent though the paramagnetic effects from oxygen are measurably lower at the tag. The reduced effect on the tag is likely a result of local decrease in hydrophobicity rather than reduced solvent exposure. For example, the ratio of the paramagnetic rate arising from dissolved oxygen to the solvent isotope shift (i.e. R1P / ΔδD2O-H2O) should give an idea of the relative hydrophobicity in the vicinity of each tyrosine. For Tyr54, Tyr49, and Tyr8, this ratio is 16.5, 18.8 and 17.8 s− 1 ppm− 1, while that associated with the tyrosine at the polyhistidine tag is 12.5 s− 1 ppm−1, implying a more hydrophilic environment, which we expect in the vicinity of the charged polyhistidine sequence. A reliable estimate of local hydrophobicity associated with Tyr10 cannot be obtained since the isotope shifts were noted above to be anomalous. Thus, we conclude based on relaxation rates in D2O, solvent isotope effects, and paramagnetic shifts and relaxation effects from dissolved oxygen that Tyr54, Tyr49, Tyr10, and Tyr8 fluorotyrosine residues are approximately equivalently and fully exposed to solvent in the absence of a binding peptide. 4.4. Effect of a binding peptide on the fluoro-Fyn SH3 domain

Fig. 5. (A) 19F NMR spectra of the metafluorotyrosine substituted Fyn SH3 domain at 10 °C in 10%, 50%, and 100% D2O. (B) 19F NMR spectra of the metafluorotyrosine substituted Fyn SH3 domain at 10 °C in H2O under ambient conditions (0.20 bar PO2) and upon equilibration at a partial pressure of 20 bar oxygen.

Tyr10. The solvent isotope shifts (ΔδD2O-H2O) associated with Tyr8, Tyr49, and Tyr54 are comparable to those observed for the fluorotyrosine label on the polyhistidine tag from which we conclude that these three residues are significantly exposed to the solvent at the meta position on the ring, while the isotope shift associated with Tyr10 is actually positive, indicating some degree of solvent protection and/or a conformational change accompanying the isotope shift. This anomalous isotope shift is not surprising since the Tyr10 chemical shift is known to vary over a range of 0.5 ppm in H2O, depending on the protein concentration. The delicate equilibrium between monomer and dimer may also be affected slightly by a substantial change in the H2O/D2O ratio. Earlier, we attributed the two upfield peaks associated with Tyr10 to a dimeric and monomeric state. At low D2O/H2O (bottom trace in Fig. 5A) the outer most peak associated with the monomer appears slightly larger. Soluble contrast agents also help to address solvent exposure. Fig. 5B shows the result of dissolved oxygen on the 19F NMR spectrum which shows significant chemical shift perturbations and line broadening. The oxygen induced shifts,

The Fyn SH3 domain is known to bind preferentially to PPII helices [51,52] with typical dissociation constants in the micromolar range, as expected with its role in cell signaling. Recently, Li et al. have employed combinatorial strategies to design peptide ligands that bind to the Fyn SH3 domain with dissociation constants nearly one thousand fold lower than those found in vivo [34]. Since the Fyn SH3 domain plays a role in T cell activation, such ligands have a therapeutic use in regulating T cell activation or altering the behavior of other Src kinases [34]. In this paper, we examine the effect on the 19F NMR spectra of the Fyn SH3 domain upon titrating an Nsubstituted peptide referred to as peptide 11a [34], having the sequence (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro– Leu–Pro–Dap–NH2, where Dap represents (L)-2,3-diaminopropionic acid. Peptide 11a binds to the wild type Fyn SH3 domain with a 110 nM dissociation constant [34]. Fig. 6A represents the 19F NMR spectra as a function of increasing concentration of peptide. Three tyrosine residues seem to be involved in a strong interaction with 11a based upon the pronounced chemical shift perturbations (i.e. Tyr8, Tyr49, and Tyr54). Tyr8, Tyr10, and Tyr54, which are known to be most conserved amongst SH3 domains also exhibit pronounced line broadening upon addition of binding peptide. Upon saturating with 11a, these residues exhibit line broadening of 36.73 Hz, 14.0 Hz, and 65.3 Hz respectively. Although Tyr10 does not exhibit a sizeable change in chemical shift upon addition of peptide, the chemical shift dependence on peptide concentration associated with Tyr54, Tyr49, and Tyr8 can be easily fitted to Eq. (1), providing a uniform estimate of the dissociation constant (i.e. 12 ± 2 μM, 22.8 ± 0.3 μM , and 19.8 ± 0.3 μM respectively, for which we assign an average dissociation

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Δν1/2, according to R2 = π × Δν1/2, where we assume each resonance is represented by a Lorenztian line. The resonances associated with Tyr54 and Tyr8 are particularly useful to evaluate the effective exchange rate, kex, since the chemical shift differences (Δω) between the free and bound state are 9050 s− 1 and 4630 s− 1, respectively. In our analysis, the line broadening data from both residues was used to obtain a global estimate of kex as a function of peptide concentration. At the maximum peptide concentration range used, the bound fraction, p1, was estimated to be 0.96, while the effective exchange rate was determined from line broadening to be 5200 ± 700 Hz (i.e. 4500 Hz and 5850 Hz for Tyr 8 and Tyr 54, respectively). 4.5. Solvent exposure of the fluorotyrosine residues in the peptide: Fyn SH3 complex

Fig. 6. (A) 19F NMR spectra of the metafluorotyrosine substituted Fyn SH3 domain at 10 °C as a function of binding peptide concentration. The Fyn SH3 domain concentration was approximately 0.4 mM in these experiments. (B) Comparison of 19F NMR spectra of the metafluorotyrosine substituted Fyn SH3 domain at 10 °C, saturated with binding peptide under ambient conditions (0.2 bar PO2) and upon equilibration at an oxygen partial pressure of 20 bar.

constant of 18 ± 4 μM). The G48M mutant has been known to increase the dissociation constant by a factor of three in studies of binding peptides with micromolar dissociation constants. Even if we attribute a factor of three to the G48M mutant, this suggests that the metafluorine labels reduce the binding efficacy to the peptide by a factor of roughly thirty. Steric perturbations resulting from the introduction of fluorine atom and consequent changes in partial charge associated with the tyrosine ring may account for the reduced binding to the peptide, while competition with dimerization may also be a factor. The line broadening seen with increasing peptide concentration as shown in Fig. 6A can be interpreted in terms of a simple two site exchange model in which the Fyn SH3 domain interconverts between a bound and free state (i.e. state 1 and 2, respectively). In the presence of peptide 11a, the equilibrium strongly favors the FynSH3 domain:11a complex (i.e. p1 H p2) in which case we make use of Eq. (2) to analyze the line broadening as a function of peptide concentration. Here, we estimate the transverse relaxation rate, R2, from the line width,

The extent of solvent interaction upon saturating the Fyn SH3 domain with the binding peptide may be reliably measured by considering the effect of dissolved oxygen. The spectra of the Fyn SH3 domain in the presence of binding peptide at either ambient oxygen concentrations or under an oxygen partial pressure of 20 bar are shown in Fig. 6B. In contrast to the measurements performed for the free protein, where all residues exhibited a comparable degree of solvent exposure, the paramagnetic shifts and relaxation rate enhancements vary significantly amongst the four fluorotyrosines, as shown in Table 1. One way of assessing the difference in solvent exposure between the free and complexed state is to consider the ratio of paramagnetic shifts or relaxation rate enhancements from dissolved oxygen, which we express as ΔδO2 (cmplx) / ΔδO2 (free) and R1P (cmplx) / R1P (free). This ratio of shifts or relaxation rates reveals that Tyr54 and Tyr10 become significantly more buried in the presence of the binding peptide, while the meta position of the other fluorotyrosines appears to undergo little change in solvent exposure. 5. Conclusions The G48M mutant of the Fyn SH3 domain was biosynthetically labeled with metafluorinated tyrosine, giving rise to 5 resonances in the 19F NMR spectra, associated with Tyr8, Tyr10, Tyr49, Tyr54 and a tyrosine on the C-terminal polyhistidine tag. Assignments were performed by comparing 19F NMR spectra with those of single phenylalanine mutations. The free protein was characterized by a monomeric and dimeric state which was suggested to arise from a fluorophobic effect. The monomeric state was found to dominate at protein concentrations of 0.4 mM or less. Solvent exposure of the various fluorinated residues was assessed by solvent isotope shift effects and by paramagnetic shifts and relaxation rates arising from dissolved oxygen at a partial pressure of 20 bar. In the free state, Tyr54, Tyr49, Tyr10, and Tyr8, in addition to the tag appear to be fully exposed to the solvent. The addition of a proline-rich peptide, (2-hydroxynicotinic-NH)–Arg–Ala–Leu–Pro–Pro–Leu–Pro-diaminopropionic acid –NH2, causes Tyr 54 and Tyr10 to be significantly less exposed to the solvent. By monitoring the chemical shift of Tyr8, Tyr49, and Tyr54 as a function of binding peptide, the

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equilibrium dissociation constant, Kd, for the interaction between the peptide and the G48M mutant of the Fyn SH3 domain, was estimated to be 18 ± 4 μM. This suggests that the introduction of metafluorinated tyrosine weakens the binding interaction with the peptide. An analysis of line widths with peptide concentration provided an estimate of the effective exchange rate, kex, between free and bound states to be on the order of 5200 Hz at a peptide concentration where 96% of Fyn SH3 is complexed with the peptide. The above experiments revealed that the resonances associated with the free and bound peptide state states were characterized by differences of 3–9 kHz which is not uncommon in 19F NMR. Traditional protein–protein interaction kinetics and equilibria are often performed by fluorescence measurements. However, in situations where the binding pocket contains one or more tyrosine residues, the above measurements demonstrate the potential utility of 19F NMR as a probe of binding. Moreover, the considerable difference between the free and bound states mean that exchange rates might be reliably measured by simple CPMG or T2 experiments for exchange processes on the submillisecond and millisecond timescale, while saturation transfer experiments should provide a reliable measure of exchange rates on the millisecond to second timescale. Acknowledgments We are grateful to professor D. S. Lawrence (Biochemistry Department, Albert Einstein College of Medicine) for kindly providing us with a peptide sample for binding studies with fluoro-Fyn SH3. This work was supported by grants from the Petroleum Research Council (R. S. Prosser), NSERC (R. S. Prosser), and CIHR (Julie Forman-Kay).

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