Probing Local Environments with the Infrared Probe - American

Feb 23, 2011 - Emily E. Smith, Barton Y. Linderman, Austin C. Luskin, and Scott H. Brewer*. Department of Chemistry, Franklin & Marshall College, Lanc...
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Probing Local Environments with the Infrared Probe: L-4-Nitrophenylalanine Emily E. Smith, Barton Y. Linderman, Austin C. Luskin, and Scott H. Brewer* Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604-3003, United States

bS Supporting Information ABSTRACT: The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro symmetric stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and 15N isotopic labeling of the three-atom nitro group of this UAA. 14NO2 and 15NO2 variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro symmetric stretching frequency of L-4nitrophenylalanine to be experimentally measured at either site in sfGFP. The 14NO2 symmetric stretching frequency red-shifted 7.7 cm-1 between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.

’ INTRODUCTION A full understanding of how proteins fold into their native three-dimensional structure is not known.1 The ability to elucidate the time-dependent structural changes associated with folding is central to solving the protein-folding problem. A number of spectroscopic techniques have been utilized to study protein conformational changes associated with protein folding, such as vibrational spectroscopy.2-7 For instance, infrared (IR) spectroscopy can distinguish between multiple protein secondary structure elements including R-helices and β-sheets by monitoring the carbonyl stretching frequency of the amide groups of the peptide backbone and has sufficient temporal resolution to probe protein-folding dynamics.8-11 IR spectroscopy has been coupled with selectively incorporated isotopic labels, such as 13Cd16O and 13Cd18O, into the amide group of amino acids to probe local structure and dynamics.9,12-21 Nitrile and azide groups have been introduced into a number of amino acid side chains to serve as sensitive infrared probes of side chain environments.22-30 For instance, the nitrile group has been incorporated into the side chain of phenylalanine,23-25 alanine,23 tryptophan,26 and cysteine,27,28 while the azide group has been incorporated into the side chain of alanine29 and phenylalanine.30 These modified amino acids have either been incorporated into peptides or proteins by solid-phase peptide synthesis,23,26,29 cyanylation of cysteine residues,27,28 or engineered orthogonal aminoacyl-tRNA synthetase/tRNA pairs.24,25,30 The genetic incorporation of unnatural amino acids (UAAs) containing spectroscopic probe(s), such as a nitrile or r 2011 American Chemical Society

azide group, is a powerful tool since this methodology can be utilized to incorporate a variety of UAAs into proteins sitespecifically for the study of protein structure and dynamics with no inherent protein size limit.30-33 Raman spectroscopy has been utilized to show that the nitro symmetric stretching frequency (~vNO2,s) of 2-nitrophenol is sensitive to solvent.34 In particular, the position of the ~vNO2,s is dependent upon both intramolecular hydrogen bonding between the hydroxyl and nitro group of 2-nitrophenol and intermolecular hydrogen bonding between the nitro group of 2-nitrophenol and solvent molecules.34 Raman spectroscopy has also illustrated the sensitivity of the ~vNO2,s of 3-nitrotyrosine to solvent.35 The ~vNO2,s of 3-nitrotyrosine blue-shifted 6 cm-1 going from the solvent hexane to water in response to hydrogen bond interactions with water molecules.35 The ~vNO2,s of 3-nitrotyrosine was also shown to be sensitive to local protein environments (extent of solvation) as probed by Raman spectroscopy in the proteins lysozyme and cytochrome c.35,36 The nitro group was introduced into these proteins by the nitration of tyrosine residue(s) in these proteins.35,36 Recently, the nitro modified phenylalanine unnatural amino acid, L-4-nitrophenylalanine,37-39 was genetically incorporated into the 64 residue bZIP protein, demonstrating that this UAA can quench the fluorescence of nearby tryptophan residues.40 Here, we demonstrate that the three-atom nitro group of L-4Received: September 28, 2010 Revised: January 27, 2011 Published: February 23, 2011 2380

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Figure 1. (A) Structure of sfGFP (PDB ID 2B3P) highlighting aspartic acid residue 134 (red) and asparagine residue 150 (blue) that were selectively replaced with either p14NO2Phe or p15NO2Phe generating four sfGFP constructs each containing one of the UAAs. The fluorescent chromophore inside the β-barrel is shown in a stick representation. (B) Structures of 14N- and 15N-labeled L-4-nitrophenylalanine.

nitrophenylalanine constitutes a sensitive IR probe of local environment. Specifically, we show that the nitro symmetric stretching frequency of L-4-nitrophenylalanine (Figure 1B) is sensitive to solvent, isotopic labeling, and different local environments in the 247 residue monomeric protein, superfolder green fluorescent protein41 (sfGFP, Figure 1A). The site-specific genetic incorporation of this UAA in response to the amber codon was achieved with high efficiency and fidelity. Isotopic labeling of the nitro group of this UAA and difference FTIR spectroscopy were employed to measure the nitro symmetric stretching frequency of L-4-nitrophenylalanine incorporated sitespecifically into sfGFP.

’ EXPERIMENTAL SECTION Materials. Chemical reagents were purchased from SigmaAldrich and Fisher Scientific and used without further purification. 15N-labeled nitric acid (98% 15N enrichment), 15N-labeled formamide (98% 15N enrichment), deuterium oxide (99.9% D enrichment), and sodium deuteroxide (99.5% D enrichment) were purchased from Cambridge Isotope Laboratories. L-4Nitrophenylalanine (p14NO2Phe, 1) and the amine-protected Boc-L-4-nitrophenylalanine (Boc-p14NO2Phe, 1a) were purchased from PepTech. 15N-labeled L-4-nitrophenylalanine (p15NO2Phe, 2) was synthesized based upon previous literature procedures with minor modifications (see Supporting Information).42 Oligonucleotides, DH10B cells, and pBadA were purchased from Invitrogen. All aqueous solutions were prepared with 18 MΩ-cm water. Expression and Purification of Superfolder GFP. The expression and purification of wild-type superfolder GFP (wtsfGFP)41 and sfGFP constructs containing either p14NO2Phe or p15NO2Phe were expressed and purified from DH10B Eschericia coli cells similar to previous literature procedures with minor modifications (see Supporting Information).33,43 Typical yields for the UAA containing sfGFP constructs was ∼0.25 g of purified protein per liter of autoinduction media containing 1 mM p14NO2Phe or p15NO2Phe. Equilibrium FTIR Measurements. Equilibrium FTIR absorbance spectra were recorded on a Bruker Vertex 70 FTIR spectrometer equipped with a globar source, KBr beamsplitter,

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and a liquid nitrogen cooled mercury cadmium telluride (MCT) detector. The spectra were the result of 1024 scans recorded at a resolution of 1.0 cm-1. The transmission measurements were recorded using a temperature-controlled cell consisting of calcium fluoride windows with a path length of either ∼25 or ∼50 μm. The ATR (attenuated total reflectance) FTIR measurements were recorded using a temperature-controlled Harrick BioATRcell II accessory. The temperature of the IR cell was controlled by a water bath and the sample temperature was measured by a thermocouple embedded in the cell. The FTIR absorbance spectra were recorded at 298 K and baseline corrected. Line Shape Fitting. Line shape analysis was used to model the IR absorbance band corresponding to the nitro symmetric stretching frequency of Boc-p14NO2Phe in THF and p14NO2Phe in H2O. The line shape function consisted of a linear combination of a Gaussian and Lorentzian function as shown in eq 1:44 " ð4 ln 2Þ1=2 Fð~ν Þ ¼ ðao þ bo ~ν Þ þ A ð1 - mLorentz Þ 1=2 π f whm e-ð4 ln 2Þð~ν - ~ν o Þ =f whm  2 f whm þ mLorentz π 4ð~ν - ~ν o Þ2 þ f whm2 2

2

ð1Þ

where mLorentz is the fraction of the Lorentzian character, ~vo is the band position, fwhm is the full-width at half-maximum for the line shape, A is the area, and (a0 þ b0~v) represents a linear baseline offset where ao is the intercept and bo is the slope. The line shape analysis was performed in Igor Pro (Wavemetrics). Density Functional Theory Calculations. Geometry optimizations and vibrational analyses were carried out on the model system nitrobenzene using the quantum chemical software package Gaussian 03 on a multiprocessor Mac Pro computer.45 The calculations were performed using the density function theory (DFT) method, the B3PW91 density functional,46,47 and a 6-31þþG(d,p) basis set.48,49 The calculations were performed in the gas phase, with or without explicit water molecules around the nitro group. The graphical user interface, GaussView 4, was utilized to build the structures and to visualize the normal modes of vibrations.

’ RESULTS AND DISCUSSION Solvent Dependence of v~NO2,s of L-4-Nitrophenylalanine. The sensitivity of the nitro symmetric stretching frequency of L4-nitrophenylalanine to solvent is illustrated in Figure 2. Figure 2 shows dependence of the ~vNO2,s of p14NO2Phe (1) in an aqueous sodium hydroxide solution and the amine-protected version of p14NO2Phe, Boc-p14NO2Phe (1a) in tetrahydrofuran (THF). Sodium hydroxide was added to the aqueous solution of 1 to enhance the solubility of 1 in H2O. The aprotic solvent THF and the protic solvent H2O were chosen to mimic the interior (solvent excluded) and exterior (solvent exposed) environments found in proteins, respectively. The IR absorbance bands corresponding to the nitro symmetric stretching frequency were fit to eq 1 to determine the change in the ~vNO2,s and full-width at halfmaximum (fwhm) for each solvent. The ~vNO2,s blue-shifted from 1347.3 cm-1 for 1a in THF to 1351.1 cm-1 for 1 in the basic aqueous solution and the fwhm of the nitro absorbance band increased from 7.6 cm-1 for 1a in THF to 9.3 cm-1 for 1 in the 2381

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Figure 2. FTIR absorbance spectra of p14NO2Phe (1) in a 100 mM aqueous sodium hydroxide solution (open circles) and Boc-p14NO2Phe (1a) dissolved in THF (open squares) in the nitro symmetric stretching region recorded at 25 °C. The spectra were fit to eq 1 (solid curves). The concentration of 1 and 1a were 50 mM. The maximum absorbance of each spectrum has been normalized to unity.

basic aqueous solution. The 3.8 cm-1 increase in the ~vNO2,s in water is principally due to hydrogen-bond interactions between the nitro group of p14NO2Phe and the water molecules in the solvent. The change in the fwhm is likely due, in part, to different solvent molecule configurations around p14NO2Phe and solvent dynamics. To explore the dependence of the ~vNO2,s on hydrogen bond interactions between the nitro group and H2O molecules, DFT calculations were carried out on a simple model system, nitrobenzene, with and without explicit water molecules in the gas phase. The nitro symmetric stretching frequency was found to blue shift 0.7 cm-1 upon the addition of one explicit water molecule in a hydrogen bonding geometry with the nitro group of nitrobenzene. The blue shift increased to 2.6 cm-1 upon the addition of two more explicit water molecules around the nitro group in a hydrogen bond geometry. The vibrational analysis was performed after geometry optimization. These simple models demonstrate that the ~vNO2,s is sensitive to hydrogen bonding with water molecules by predicting that this interaction results in a blue shift in the ~vNO2,s, which is in qualitative agreement with the experimental results. These results also show that the magnitude of this shift is dependent upon the number of water molecules and their geometry relative to the nitro group (see Supporting Information for structures). The calculated blue shift upon hydrogen bonding with water molecules is less than the experimental frequency shift. This difference is principally due to the simplicity of the model, the theoretical method employed, and the calculations being performed in the gas phase. However, these results do support that the origin of the experimentally measured shift in the ~vNO2,s upon going from THF to water is at least, in part, due to hydrogen bond interacts between the nitro group of 1 and water molecules of the solvent. Isotope Effect on v~NO2,s of L-4-Nitrophenylalanine. The impact of 15N labeling of the NO2 group of p14NO2Phe on the nitro symmetric stretching frequency is illustrated in Figure 3. Figure 3A shows the IR absorbance band corresponding to the nitro symmetric stretching frequency of p14NO2Phe (1) and p15NO2Phe (2) in a basic aqueous solution. The nitro symmetric stretching frequency red shifts from 1351.1 to 1326.9 cm-1 upon incorporation of the 15N isotopic label in the nitro group. This 24

Figure 3. (A) FTIR absorbance spectra of p14NO2Phe (1, solid curve) and p15NO2Phe (2, dashed curve) in a basic aqueous solution in the nitro symmetric stretching region recorded at 25 °C. The concentration of 1 and 2 were 50 mM. The maximum absorbance of each spectrum has been normalized to unity. (B) Corresponding difference FTIR spectrum formed by subtracting the FTIR absorbance spectrum of 2 from the spectrum of 1.

cm-1 isotopic shift is highlighted in the difference FTIR absorbance spectrum formed from the subtraction of the FTIR absorbance spectrum of 2 from the absorbance spectrum of 1 (Figure 3B). The difference spectrum results in the subtraction of the absorbance of any vibrations not involving motion of the nitrogen atom of the nitro group. Thus, the difference spectrum aids in the isolation and identification of the nitro symmetric stretching frequency of 1 and 2. To confirm the origin of the positive and negative features in the difference spectrum shown in Figure 3B, DFT was utilized to calculate the effect of 15N isotopic labeling on the nitro symmetric stretching frequency. Gas phase DFT calculations predicted that the 15 N labeling of the nitro group of the geometry optimized model nitrobenzene resulted in a 24 cm-1 red shift in agreement with the experimental results in both magnitude and direction. These computational results are also in agreement with previous DFT calculations of the 15N isotopic shift of the ~vNO2,s in nitrobenzene.50 Therefore, the positive feature at 1351.1 cm-1 in Figure 3B is due to the 14NO2 symmetric stretch of 1, while the negative feature at 1326.9 cm-1 is due to the 15NO2 symmetric stretch of 2. The combination of difference FTIR spectroscopy and 15N isotopic labeling of the nitro group significantly aids in the ability of the nitro symmetric stretching frequency to be an effective infrared probe of local protein environments as demonstrated below. 2382

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Sensitivity of v~NO2,s to Local Protein Environments in sfGFP. The sensitivity of the nitro symmetric stretching frequency

Figure 4. Difference FTIR spectra formed by the subtraction of the FTIR absorbance spectra of sfGFP-134-p15NO2Phe and sfGFP-134p14NO2Phe (A) or sfGFP-150-p15NO2Phe and sfGFP-150-p14NO2Phe (B) in the nitro symmetric stretching region recorded at 25 °C in a pH 7.3 aqueous buffer containing 50 mM sodium phosphate and 150 mM sodium chloride. In each case the FTIR absorbance spectrum of sfGFPp15NO2Phe was subtracted from the absorbance spectrum of sfGFPp14NO2Phe. The concentration of the protein constructs were ∼1 mM.

Structure of Superfolder Green Fluorescent Protein. sfGFP is a monomeric, 247 residue protein consisting of 47% β-sheet and 10% helical structure as shown in Figure 1A. The unnatural amino acids p14NO2Phe and p15NO2Phe were separately incorporated at site 134 in sfGFP (sfGFP-134-p14NO2Phe and sfGFP-134-p15NO2Phe, respectively), which is in a solvent exposed loop region of the protein (shown in red in Figure 1A). p14NO2Phe and p15NO2Phe were also separately incorporated at position 150 in sfGFP (sfGFP-150-p14NO2Phe and sfGFP-150p15NO2Phe, respectively) shown in blue in Figure 1A, which is in a β-sheet region of the β-barrel of the protein that is partially buried (desolvated) by neighboring amino acid side chains. The nature of the two distinct environments represented by site 134 and 150 in sfGFP were further characterized by calculating the solvent accessible surface area of these sites using the software GETAREA.51 The solvent accessible surface area (SASA) for D134 and N150 in wild-type sfGFP using a probe radius of 1.4 Å is 100 Å2 and 55 Å2, respectively. For comparison, the SASA of fully solvated aspartic acid and asparagine in the random coil tripeptide Gly-Asp-Gly and Gly-Asn-Gly is 113 Å2 and 114 Å2, respectively. Therefore, site 134 represents a solventexposed region of the protein while site 150 is partially buried (desolvated).

of L-4-nitrophenylalanine to local protein environments in sfGFP is shown in Figure 4. Figure 4A shows the difference FTIR spectrum corresponding to the subtraction of the FTIR absorbance spectrum of sfGFP-134-p15NO2Phe from the absorbance spectrum of sfGFP-134-p14NO2Phe in the nitro symmetric stretching region. This subtraction results in a positive band at 1352.3 cm-1 and a negative band at 1328.2 cm-1, which correspond to the ~vNO2,s of p14NO2Phe and p15NO2Phe incorporated at site 134 in sfGFP, respectively. The isolation of the nitro symmetric stretch IR absorbance band for both UAAs is possible since any potentially interfering protein IR absorbance bands not involving the nitrogen atom of the nitro group are removed in the formation of the difference spectrum. This removal is effective since the 15N isotopic label, which is the only difference between the two protein constructs, is minimally perturbative to the structure of the protein (see Supporting Information). The identification of the IR absorbance bands in Figure 4A is significantly aided by isotopic labeling of the nitro group with 15 N. The identification of the bands, as corresponding to the nitro symmetric stretch, is due to both the absolute positions of the bands and the relative separation between the two observed bands. First, the position of the band is similar to the position of the IR nitro absorbance bands of free p14NO2Phe and p15NO2Phe in a basic aqueous solution (see Figure 3). Second, the experimentally observed isotopic shift of 24 cm-1 for p14NO2Phe and p15NO2Phe incorporated at position 134 in GFP is consistent with the experimentally measured isotopic shift of the nitro stretching frequency for the free UAAs in aqueous solution (see Figure 3) and the DFT calculated isotopic shift for nitrobenzene. The observed frequency of the ~vNO2,s of p14NO2Phe incorporated at site 134 in sfGFP confirms that this position of the protein is solvent exposed due to the similarity of this frequency with the ~vNO2,s of free p14NO2Phe in a basic aqueous solution as expected based upon the structure of sfGFP. Figure 4B shows the corresponding difference FTIR spectrum generated by the subtraction of the FTIR absorbance spectrum of sfGFP-150-p15NO2Phe from the absorbance spectrum of sfGFP150-p14NO2Phe. Similar to Figure 4A, this difference spectrum contains one positive and one negative band; however, the positions of these bands are shifted to 1344.6 and 1321.0 cm-1, respectively, in Figure 4B. The positive feature corresponds to the ~vNO2,s of p14NO2Phe, while the negative feature corresponds to the ~vNO2,s of p15NO2Phe incorporated at site 150 in sfGFP. This assignment is based upon the comparison of the position of the nitro symmetric stretch IR absorbance bands of 1 and 1a in a basic aqueous solution and THF, respectively, and upon the similarity of the observed isotopic shift (24 cm-1) with the isotopic shift measured for 1 and 2 in a basic aqueous solution (see Figures 2 and 3). The position of the nitro symmetric stretch IR absorbance band for sfGFP-150-p14NO2Phe illustrates that p14NO2Phe at position 150 in sfGFP is at least partially buried from solvent for two reasons. First, the ~vNO2,s of sfGFP-150-p14NO2Phe is red-shifted by 7.7 cm1 compared to the~vNO2,s of sfGFP-134-p14NO2Phe, which is similar in direction to the red shift of the ~vNO2,s for Boc-p14NO2Phe in THF compared to free p14NO2Phe in a basic aqueous solution. This red shift is indicative of a decrease in hydrogen bond interactions between the nitro group of p14NO2Phe and water molecules of the solvent. Second, the position of the ~vNO2,s of sfGFP-150-p14NO2Phe is closer in frequency to Boc-p14NO2Phe 2383

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The Journal of Physical Chemistry B in THF than free p14NO2Phe in a basic aqueous solution. The classification of site 150 as a partially buried site based upon the ~vNO2,s of sfGFP-150-p14NO2Phe and sfGFP-150-p15NO2Phe is expected based upon the structure of sfGFP. The results for p14NO2Phe and p15NO2Phe incorporated at either site 134 or site 150 in sfGFP illustrate the ability of the nitro symmetric stretch of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments due to the experimentally measured shift in the ~vNO2,s between the two sites. The isolation and identification of the nitro symmetric stretch IR absorbance band resulted from the combination of isotopic labeling of the nitro group and difference FTIR spectroscopy.

’ CONCLUSIONS The nitro symmetric stretching frequency of L-4-nitrophenylalanine was shown to be a sensitive IR probe of local environment. The ~vNO2,s blue-shifted from 1347.3 cm-1 for Bocp14NO2Phe in THF to 1351.1 cm-1 for p14NO2Phe in a basic aqueous solution. This shift was principally due to the formation of hydrogen bonds between the water molecules of the solvent and the nitro group of p14NO2Phe as confirmed by DFT calculations. Incorporation of a 15N isotopic label in the nitro group of L-4-nitrophenylalanine resulted in a 24 cm-1 red shift in the ~vNO2,s in agreement with the DFT calculated isotopic shift in both magnitude and direction. The ability of the ~vNO2,s to effectively probe different local protein environments was confirmed by the incorporation of either p14NO2Phe or p15NO2Phe into sfGFP in a solvent exposed (site 134) or a partially buried position (site 150) utilizing an engineered orthogonal aminoacyltRNA synthetase/tRNA pair with high efficiency and fidelity. Isotopic labeling and difference FTIR spectroscopy easily allowed isolation and identification of the nitro symmetric stretch IR absorbance band in the protein constructs containing either UAA. The ~vNO2,s of p14NO2Phe red-shifted 7.7 cm-1 between site 134 and 150, as expected, based upon the nature of each site in the protein and the observed shifts of the ~vNO2,s of 1 in H2O and 1a in THF. These results therefore verify the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments. The sensitivity of the nitro symmetric stretching frequency of L-4-nitrophenylalanine coupled with the temporal resolution of IR spectroscopy and the lack of a protein size limit for the genetic incorporation of this UAA results in an effective methodology for the study of protein conformational changes associated with protein folding with site-specificity. ’ ASSOCIATED CONTENT

bS15

Supporting Information. Synthetic procedure for p NO2Phe, 1H and 15N NMR spectra of p15NO2Phe, protein expression and purification procedure, SDS-PAGE gel of the protein constructs, ESI-Q-TOF mass spectra of the proteins, ATR-FTIR spectra of the sfGFP constructs, sequence of wildtype sfGFP, and DFT optimized structures of nitrobenzene with and without explicit water molecules including the eigenvector projections for the nitro symmetric stretch. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Telephone: (717) 358-4766. Fax: (717) 291-4343.

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’ ACKNOWLEDGMENT This work was supported by F&M Hackman and Eyler funds. We thank Ryan Mehl, Kenneth Hess, and Beth Buckwalter for their technical assistance and gratefully acknowledge the help of Lisa Mertzman and Carol Strausser. ’ REFERENCES (1) Dill, K. A.; Ozkan, S. B.; Weikl, T. R.; Chodera, J. D.; Voelz, V. A. Curr. Opin. Struct. Biol. 2007, 17, 342–346. (2) Bunagan, M. R.; Gao, J. M.; Kelly, J. W.; Gai, F. J. Am. Chem. Soc. 2009, 131, 7470–7476. (3) Montalvo, G.; Waegele, M. M.; Shandler, S.; Gai, F.; DeGrado, W. F. J. Am. Chem. Soc. 2010, 132, 5616–5618. (4) Balakrishnan, G.; Weeks, C. L.; Ibrahim, M.; Soldatova, A. V.; Spiro, T. G. Curr. Opin. Struct. Biol. 2008, 18, 623–629. (5) Mikhonin, A. V.; Asher, S. A. J. Am. Chem. Soc. 2006, 128, 13789–13795. (6) Brewer, S. H.; Vu, D. M.; Tang, Y. F.; Li, Y.; Franzen, S.; Raleigh, D. P.; Dyer, R. B. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16662–16667. (7) Religa, T. L.; Johnson, C. M.; Vu, D. M.; Brewer, S. H.; Dyer, R. B.; Fersht, A. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9272–9277. (8) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469–487. (9) Decatur, S. M. Acc. Chem. Res. 2006, 39, 169–175. (10) Ganim, Z.; Chung, H. S.; Smith, A. W.; Deflores, L. P.; Jones, K. C.; Tokmakoff, A. Acc. Chem. Res. 2008, 41, 432–441. (11) Callender, R.; Dyer, R. B. Chem. Rev. 2006, 106, 3031–3042. (12) Huang, C. Y.; Getahun, Z.; Zhu, Y. J.; Klemke, J. W.; DeGrado, W. F.; Gai, F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 2788–2793. (13) Torres, J.; Briggs, J. A. G.; Arkin, I. T. J. Mol. Biol. 2002, 316, 365–374. (14) Werner, J. H.; Dyer, R. B.; Fesinmeyer, R. M.; Andersen, N. H. J. Phys. Chem. B 2002, 106, 487–494. (15) Fang, C.; Wang, J.; Charnley, A. K.; Barber-Armstrong, W.; Smith, A. B.; Decatur, S. M.; Hochstrasser, R. M. Chem. Phys. Lett. 2003, 382, 586–592. (16) Fang, C.; Wang, J.; Kim, Y. S.; Charnley, A. K.; BarberArmstrong, W.; Smith, A. B., III; Decatur, S. M.; Hochstrasser, R. M. J. Phys. Chem. B 2004, 108, 10415–10427. (17) Huang, R.; Kubelka, J.; Barber-Armstrong, W.; Silva, R. A. G. D.; Decatur, S. M.; Keiderling, T. A. J. Am. Chem. Soc. 2004, 126, 2346–2354. (18) Mukherjee, P.; Krummel, A. T.; Fulmer, E. C.; Kass, I.; Arkin, I. T.; Zanni, M. T. J. Chem. Phys. 2004, 120, 10215–10224. (19) Brewer, S. H.; Song, B. B.; Raleigh, D. P.; Dyer, R. B. Biochemistry 2007, 46, 3279–3285. (20) Kim, Y. S.; Liu, L.; Axelsen, P. H.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 17751–17756. (21) Shim, S. H.; Gupta, R.; Ling, Y. L.; Strasfeld, D. B.; Raleigh, D. P.; Zanni, M. T. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 6614–6619. (22) Lindquist, B. A.; Furse, K. E.; Corcelli, S. A. Phys. Chem. Chem. Phys. 2009, 11, 8119–8132. (23) Getahun, Z.; Huang, C. Y.; Wang, T.; De Leon, B.; DeGrado, W. F.; Gai, F. J. Am. Chem. Soc. 2003, 125, 405–411. (24) Schultz, K. C.; Supekova, L.; Ryu, Y.; Xie, J.; Perera, R.; Schultz, P. G. J. Am. Chem. Soc. 2006, 128, 13984–13985. (25) Taskent-Sezgin, H.; Chung, J.; Patsalo, V.; Miyake-Stoner, S. J.; Miller, A. M.; Brewer, S. H.; Mehl, R. A.; Green, D. F.; Raleigh, D. P.; Carrico, I. Biochemistry 2009, 48, 9040–9046. (26) Waegele, M. M.; Tucker, M. J.; Gai, F. Chem. Phys. Lett. 2009, 478, 249–253. (27) Fafarman, A. T.; Webb, L. J.; Chuang, J. I.; Boxer, S. G. J. Am. Chem. Soc. 2006, 128, 13356–13357. (28) McMahon, H. A.; Alfieri, K. N.; Clark, C. A. A.; Londergan, C. H. J. Phys. Chem. Lett. 2010, 1, 850–855. (29) Oh, K. I.; Lee, J. H.; Joo, C.; Han, H.; Cho, M. J. Phys. Chem. B 2008, 112, 10352–10357. 2384

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