J . Am. Chem. SOC.1992, 114, 7463-7469
7463
Cysteine Conformation and Sulfhydryl Interactions in Proteins and Viruses. 2. Normal Coordinate Analysis of the Cysteine Side Chain in Model Compoundsf Huimin Li,’ Charles J. Wurrey,! and George J. Thomas, Jr.*.* Contribution from the Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 641 10. Received December 23, 1991 Abstract: We report vibrational normal mode analyses of cysteine and several model mercaptans containing the -CHRCH$H group (R = H or CH3). The results provide a basis for assigning observed Raman frequencies to specific conformers of the cysteinyl C,-CrS-H side chain and for a w i n g SH hydrogen bonding with appropriate donor and acceptor groups. Vibrational spectra and normal coordinate analyses of L-cysteine, 1-propanethiol (lPT), 1-propanedeuteriothiol (lPT-dl), and 2methyl-1-propanethiol (2MlPT) and corresponding experimental data on 2-methyl-2-propanethiol derivatives (2M2PT and 2M2PT-dl) indicate that conformation-sensitive modes occur in both the C-S stretching (600 < ucs < 800 cm-l) and the S-H stretching (2500 < usH < 2650 cm-I) regions of the Raman spectrum. Using a generalized valence force field, we have correlated the side-chain torsion angle (XI) of the C,-CB bond with u a , and the torsion angle (A?) of the CB-S bond with both ucs and uSH. The principal conclusions from the present study are as follows: (i) The force field obtained from 1PT is satisfactorily transferable to 2MlPT and L-cysteine and reproduces the observed trends for USH and ucs of these molecules. (ii) ucs can be shifted substantially (up to 50 cm-’) by changes in XI, but is less sensitive ( < l o cm-l) to changes in A?. (iii) uSHis perturbed sufficiently by changes in A? to account for previously reported frequency differences in Raman S H bands of cysteinyl gauche rotamers.’ Correlations are proposed for the dependence of both ucs and uSH upon conformation of the C,-CrS-H network. These correlations are expected to be useful for determining cysteine sidechain environments in proteins and their assemblies, including virions and viral precursors.
Introduction The thiol group ( S H ) of cysteine is capable of donating and accepting intramolecular hydrogen bonds which contribute to the stabilization of native protein ~tructures.”~ A recent survey6 of crystallographic structures in the Brookhaven Protein Data Bank indicates that 72% of cysteine SH groups participate in S-H-.O or S-H-N contacts of less than 4 A. In the majority of cases (62%) the acceptor is a carbonyl oxygen. About half of all cysteines are located in domains of a-helical secondary structure, in which the SH group a t position i is capable of donating a hydrogen bond to the peptide carbonyl of residue i-4, provided that the side-chain Ca-CBtorsion (XI, Figure 1) is approximately d o o (gauche- rotamer). Similarly, cysteine residues just beyond the C-terminus of an a-helix can form a side-chain to main-chain hydrogen bond, supplanting a main-chain NH..43=C linkage and thus serving to “cap“ the ~x-helix.~.’The distribution of cysteine residues in proteins with respect to the side-chain CB-S torsion (A?,Figure 1) has also been studied, indicating a preference for the gauche+ range (60-80°).3 Collectively, these analyses reveal a variety of rotamer conformations and hydrogen-bonding environments accessible to the cysteine side chain, including configurations which may play a role in defining boundaries of a-helix formation during protein folding. Cysteine side-chain conformations and hydrogen-bonding interactions in selected proteins have been assessed by surveying neutron and X-ray crystallographic data.6 However, other methods are required to probe cysteine interactions which are defined only for noncrystalline proteins, including aqueous protein solutions. Examples are the thiols of membrane proteins, which act as intra- or intercellular antioxidants by scavenging free radicals: and the reduced forms of thioredoxin and glutaredoxin, which serve ubiquitously as protein disulfide reductases? Raman spectroscopy offers several advantages for probing such cysteine interactions, including its capability for detecting SH Raman markers in both solution and crystal structures, its suitability for monitoring deuterium exchange dynamics of the SH group, and its relatively rapid data collection and analysis protocols. ~
*Author to whom correspondence may be addressed. +PartXXXVI in the series Structural Studies of Viruses by Laser Raman Spectroscopy; supported by NIH Grant AI1 1855 (G.J.T.). *Divisionof Cell Biology and Biophysics, School of Biological Sciences. I Department of Chemistry, College of Arts and Sciences.
0002-786319211514-7463$03.00/0
Recently, we reported a correlation of the Raman S-H stretching frequency (uSH) with hydrogen-bonding donor and acceptor interactions of the thiol group in cysteine model compounds.’ Other investigators have noted the sensitivity of the Raman C$ stretching frequency (uCs) to conformation of the cysteine side In the present work, we report Raman spectra of normal and deuterated cysteine model compounds and describe normal coordinate calculations performed on L-cysteine, 1-propanethiol (lPT), 1-propanedeuteriothiol ( l P T - d J , and 2methyl-1-propanethiol (2M lPT), using a refined valence force field. The results indicate correlations of the cysteine side-chain torsion XI with the Raman C$ stretching frequency, and of the torsion 2 (Figure 1) with both u a and USH. These correlations have not been recognized previously. The present study extends earlier work by providing a basis for understanding the conformational dependence of Raman S-H and C-S stretching vibrations of the cysteine moiety. The results are expected to facilitate structural interpretation of Raman bands assigned to cysteine residues of proteins. Additionally, these results should be useful for interpreting changes observed in Raman SH bands of viral proteins in different assembly states.’*J3 Materials and Methods 1. Materials. The mercaptans 1-propanethiol(1PT) and 2-methyl1-propanethiol (2MlPT) were purchased from Aldrich. 1-Propane(1) Li, H.;Thomas, G. J., Jr. J. Am. Chem. Soc. 1991, 113, 45-62. (2) Kollman, P.; McKelvey, J.; Johansson, A.; Rothenberg, S.J. Am. Chem. Soc. 1975.97, 955-965. (3) Ippolito, J. A.; Alexander, R. S.;Christianson, D. W. J . Mol. Biol. 1990,2i5,457-471. (4) Burley, S.K.; Petsko, G. A. Ado. Protein Chem. 1988,39, 125-189. ( 5 ) McGrath, M. E.; Wilke. M. E.; Higaki, J. N.; Craik, C. S.;Fletterick, R. J..Biochemistry 1989, 28,9264-9270. (6) Gregoret, L. M.; Rader, S. D.; Fletterick, R. J.; Cohen, F. E.Proteins: Struct. Funcr. Genet. 1991, 9, 99-107. (7) Presta, L. G.; Rose, G. D. Science 1988, 240, 1632-1641. (8) Frei, B.; Stocker, R.; Ames, B. N. Proc. Natl. Acad. Sci. U S A . 1988, 85,9748-9752. (9)Holmgren, A. J. Biol. Chem. 1989, 264, 13963-13966. (10)Kuptsov, A. H.; Trofimov, V. I. J . Biomol. Srruct. Dyn. 1985, 3, 185-196. (11) (a) Nogami, N.; Sugeta, H.; Miyazawa, T. Chem. Lett. 1975, 147-150. (b) Nogami, N.; Sugeta, H.; Miyazawa, T. Bull. Chem. Soc. Jpn. 1975, 48,2417-2420. (12)Thomas,G. J., Jr.; Li, Y.; Fuller, M. T.; King, J. Biochemistry 1982, 21,3866-3878. (13)Li, T.;Chen, 2.;Johnson, J. E.; Thomas, G. J., Jr. Biochemistry 1990, 29, 5018-5026.
0 1992 American Chemical Society
1464 J . Am. Chem. SOC.,Vol. 114, No. 19, 1992
Li et al. Table I. Raman Frequencies and Assignments of 1-Propanethiol and
X2
+H
(A,
H
SH
SH
H
*H
H
I
H
H
I
H
I
Figure 1. (A) Torsion angles of the cysteine side chain. (B) Internal coordinates of the cysteine C, linkages. (C) Rotamers IPH,2 P ~'Pc, , and 2 P of~ 1-propanethiol. PH designates the thiol hydrogen in the trans orientation with respect to a hydrogen substituent on the adjoining methylene carbon. Pc designates the trans orientation of the methyl carbon with respect to SH. The superscript prefm (1 or 2) indicates the relevant torsion angle (XIor A?), as well as the number of bonds separating the dihedral linkage from the peptide carbon (CJ
1-Propanedeuteriothiol 1-DroDanethiol . . frequency assignment (IPH) 2967 CH, stretch 2967 CHJ stretch 2932 CH2 stretch 2932 CH2 stretch 2874 CH2 stretch 2874 CH2 stretch 2856 CH, stretch 2589" S-H stretch (2Pc) 2581" S-H stretch 1461 CH2 scissor 1453 CH, deformation 1453 CH, deformation 1435 CH2 scissor 1381 CH, deformation 1353 CH2 wagging ('Pc) 1336 CHI wagging 1295 CH2 wagging 1248 CHI twist 1217 CH2 twist 1109 C C stretch 1088 CHI rocking (IPc) 1068 CH, rocking 1034 C-C stretch 923 CH3 rocking 896 CH2 rocking 876 CH2 rocking ('Pc) 816 C-S-H bend 792 C-S-H bend ('Pc) 772 CHI rocking 732 CH2 rocking ('Pc) 704 C-S stretch (IPc) 650 C-S stretch
1-~ro~anedeuteriothio1 . . frequency assignment (IPH) 2964 CH, stretch 2964 CHJ stretch 2933 CH2 stretch CHI stretch 2933 2874 CH2 stretch 2874 CHI stretch 2856 CH3 stretch 1876" 1457 1457 1457 1434 1379 1353 1334 1294 1244 1213 1098 1082 1058 1033 899 890 854
S-D stretch CH2 scissor CH, deformation CH3 deformation CHI scissor CH3 deformation CH2 waggfng ( ' P d CHI wagg!ng CH2 wagging CHI twist CH2 twist C C stretch CH, rocking ('PC) CHp rocking C C stretch CH, rocking CH2 rocking CH2 rocking ('Pc)
793 730
CH2 rocking CH2 rocking ('PC)
655 C-S stretch 617 C-S-D bend 412 413 C C C bend C C - C bend 361 361 C - C C bend (IPC) C - C C bend ('Pc) 284 282 C C - S bend C-C-S bend 240 240 CH, torsion CH3 torsion 190b C-S torsion 150b C-S torsion 137 C C torsion 120 C-C torsion "Data from 1% 1PT and 1% lPT-dI solutions (mol/mol) in CC4. Data from ethanethiol and ethanedeuteriothiol (ref 28).
deuteriothiol (lPT-d], 95%) and 2-methyl-2-propanedeuteriothiol (2M2PT-dl, 95%) were prepared by stirring 1PT and 2M2PT in D 2 0for 48 h at room temperature. All mercaptans were freshly distilled and chromatographed on a 15-cm column of baked alumina gel before use to remove traces of water or polar contaminants. The absence of impurities containing hydroxyl or amino groups was verified by Raman or infrared spectroscopy, as described.14 Orthorhombic L-cysteine was obtained from Sigma, and its crystal s t r u c t ~ r ewas ' ~ verified by X-ray diffraction (courtesy of Professor A. H.-J. Wang, University of Illinois, Urbana-Champaign). 2. Spectroscopy. Raman spectra were excited in the 90" scattering geometry with the 514.5-nm line of a Coherent Innova-70argon ion laser, using approximately 100 mW of radiant power at the sample. Samples were sealed in glass capillary tubes (Kimax #34507) maintained at ambient temperature (25 "C). Spectra in the interval 100-3500 cm-l were recorded on a Spex Ramalog 1401 spectrometer under the control of an IBM microcomputer. Data were collected at intervals of either 0.5 or 1.0 cm-l, with an integration time of 1.0 s and spectral slit width of 1 cm-I. Frequencies, listed in Table I, were calibrated using indene as
a standard. Reported S-H and C-S group frequencies are accurate to within 1 cm-' for strong or sharp bands. FTIR data were obtained from 400 to 4000 cm-' with a Mattson Instruments Sirius 100 spectrometer. Usually, 800 scans each of the liquid sample and background were collected with spectral resolution of 8 cm-' and a triangular apodization function. Liquids were contained in a variable-thickness cell with CaF2 windows. Spectra of lPT, 2PT, ZMlPT, and 2M2PT in the gas phase were obtained from the chromatographically pure liquids distilled at their ambient vapor pressure ( 35 cm-I), in part because of the change in major rotamer from 'PH in 1PT to 'Pc in 2M1PT.33 Additional interaction force constants [f(49), f(50),A51), andA52)] are required to improve agreement between experimental and calculated frequencies to an average error of 15 cm-I, as given in Table 111. With the additional force constants, the 'PH to 'Pc conformation change of 2 M l P T increases the calculated ucs by 49 cm-I, which approximates quite well the observed increase of 45 cm-I. The assignments for other vibrational modes are in good agreement with those for l-chloro-2methylpr~pane.'~ The better agreement achieved between the observed and calculated ucs values of 1PT is attributed to its lack of a C, methyl substituent which significantly simplifies the corresponding force field. For 2MlPT, the correlation of ucs with XI is also plotted in Figure 4. Neglect of the above noted interaction force constants elevates the bCsfrequencies less than 15 cm-l and does not change the shape of the Figure 4 plot. The calculated dependence of ucs on $ is marginal (
