Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4025-4029, April 1996 Chemistry Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel (polypeptides/s values) KATRIN GROEBKE, PETER RENOLD, KWOK YIN TSANG, THOMAS J. ALLEN, KIM F. MCCLURE, AND D. S. KEMP* Department of Chemistry, Room 18-582, Massachusetts Institute of Technology, Cambridge, MA 02139 Communicated by Julius Rebek, Jr., Massachusetts Institute of Technology, Cambridge, MA, December 27, 1995 (received for review September 26, 1995) ABSTRACT The helicity in water has been determined for 1A, but as previously noted by Scheraga and coworkers (15, 16) several series of alanine-rich peptides that contain single as well as by others (17, 18), it may also adopt conformations lysine residues and that are N-terminally linked to a helix- in which the lysine side chain packs against the helix barrel inducing and reporting template termed Ac-Hel1. The helix- (Fig. 1B). In the latter, lysine may stabilize the helix through propagating constant for alanine (SAla value) that best fits the a position-dependent electrostatic interaction between the properties ofthese peptides lies in the range of 1.01-1.02, close NH3 ion and the helix dipole (9, 15, 19, 20), through a ,r-type to the value reported by Scheraga and coworkers [Wojcik, J., hydrogen bond between the NH3 ion and a carbonyl oxygen Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, of the helix core, and through hydrophobic contacts between 121-134], but significantly lower than the value assigned by the hydrocarbon portion of the lysine side chain and the helix Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & core (13, 21). If lysine-bearing helices are significantly stabi- Baldwin, R. L. (1994) Protein Sci. 3, 843-852]. From a study lized by charge-dipole interactions, their helicity is expected of conjugates Ac-Heli-Alan-Lys-Alam-NH2 and analogs in to be sensitive to the position of the lysine along the peptide which the methylene portion of the lysine side chain is sequence. Moreover, for conformations like that of Fig. 1B, the truncated, we find that the unusual helical stability ofAlanLys stabilization attributable to hydrophobic contacts, charge- peptides is controlled primarily by interactions of the lysine dipole effects, and Tr-hydrogen bonding must change if the side chain with the helix barrel, and only passively by the length of the lysine side chain is shortened. alanine matrix. Using 'H NMR spectroscopy, we observe We have explored this issue in three ways. The tic values for nuclear Overhauser effect crosspeaks consistent with proton- template-peptide conjugates of structure Ac-Hell-Alan-Lys- proton contacts expected for these interactions. Alam-NH2 have been used to assign SAla and to measure the sensitivity ofSLys to position. The t/c changes that result when The unusually stable helices formed in water by (Ala4-Lys)n lysine is replaced by amino acid analogs bearing shorter polypeptides and their analogs have received much recent methyleneammonium functions -(CH2)n-NH3, n = 1-3 have attention (1-7), but the cause of their stability remains con- been used to probe the dependence of helicity on the posi- troversial (8, 9). Helical stability can be easily measured for the tioning of positive charge along the amino acid side chain. helices induced in short polypeptides that are N-terminally Nuclear Overhauser effects (NOEs) from 'H NMR spectra of linked to Ac-Hell (Fig. 1C), and these conjugates can be used suitably deuterated Ac-Heli-Alan-Lys-Alam-NH2 derivatives to distinguish stabilizing effects arising from initiation of a have provided structural evidence for the presence of compact helix from those attributable to its propagation (10-13). As a conformations such as that shown in Fig. 1B. capping group at the N terminus of a polypeptide, Ac-Hell acts as a helix initiator, and the stability of the helices it initiates MATERIALS AND METHODS within the peptide is proportional to the trans/cis value (t/c) of Ac-Hell, measurable as a ratio of intensities of 'H NMR Peptide conjugates were synthesized by solid-phase synthesis resonances assigned to s-trans and s-cis conformers of its using Knorr resin (Advanced ChemTech) following standard acetamido function (12, 13). For a particular amino acid 9-fluorenylmethoxycarbonyl t-butoxycarbonyl (Fmoc)/(Boc) residue the helix propagation constant s measures its tendency protocols; the conjugates were purified by preparative HPLC to join a linked, preexisting helix (14), and for a series of on a Waters RCM C18 reversed-phase column, and the purity template-linked peptides such as Ac-Hel,-Xn-NH2 or Ac-Hell- (>99%) was assessed by examination of distinctive Ac-Hell Y-Xn-NH2, where Y represents any linking peptide, an average 'NMR resonances. All peptide conjugates were characterized sx can be assigned to amino acids in the X, region from the by 'NMR and by plasma desorption mass spectroscopy as curvature of a plot of tic vs. n (10, 11, 13). Although one can described (12). All structures shown in Fig. 1 were produced argue for differences between the helices of this study, prop- using the program QUANTA (Molecular Simulations, Burling- agated unidirectionally in the N-to-C direction, and helices ton, MA). that are propagated from the C terminus or propagated NMR spectra were recorded on a Varian VXR-500S spec- bidirectionally, until evidence to the contrary is forthcoming, trometer in 2H20 solution. Integration ratios were measured it is simpler to view propagation effects measured at a distance as reported (12). For the series studied, spectra were found to from the initiation site as insensitive to the site or nature of the be invariant to dilution. For the rotating-frame Overhauser initiation. effect spectroscopy (ROESY) spectra (22, 23), a 3.3-kHz A lysine residue at the C terminus or in the core of an a-helix pulsed-spin lock was applied during the 400-ms mixing time. may adopt the solvent-exposed conformation shown in Fig. The spin lock field was generated by a train of 30° pulses (24). The carrier frequency was set downfield to the residual water The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviation: NOE, nuclear Overhauser effect; trans/cis, t/c. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 4025 Downloaded by guest on September 29, 2021 4026 Chemistry: Groebke et al. Proc. Natl. Acad. Sci. USA 93 (1996) 0 N 0

I +

N6' ,

A

(helical)

C FIG. 1. Structural models of helical Ac-Hell-Ala5-Lys-Ala2-NH2 in water; the conformation ofthe peptide backbone is consistent with the helical structure assigned to Ac-Hell-Ala6-OH by NMR analysis (10-13). (A) The lysine side chain is shown in a solvent-exposed conformation (approximate angles: Xl = g-; all other X = t). (B) The lysine side chain adopts a solvent-shielded (compact) conformation with approximate angles: Xi, X2, X3, X4 = t, X5 = g-. (C) The helicity-inducing template Ac-Hell in the helix-nucleating te-state and the nonnucleating cs-state. In a c-state (cis) the CH3-CO-N of Ac-Hell adopts the proline cis conformation; in a t-state (trans) the CH3-CO-N dipole is aligned with the helix dipole and can form hydrogen bonds with amides at the helix N terminus as shown in A and B. For peptide conjugates of Ac-Hell in water, only a t-state is found to initiate helices, and a reorientation of the sulfur (s -> e) accompanies the initiation, leading to the te-state. A peptide associated with Ac-Hell in the cs-state has a random coil structure (10, 12). (An additional nonnucleating conformation, ts, is demonstrable but not depicted.) Slow rotation about the CH3-CO-N bond results in separate resonances for t- and c-states in 'H NMR spectra of Ac-Hell derivatives. The fractional helicity of a template-linked peptide has been shown to be directly correlated to the fraction of template in the te-state and can be calculated from t/c = ([ts] + [te])/[cs], with t/c representing the ratio between the integrated intensities of t-state and c-state NMR resonances (10, 13). peak to suppress homonuclear Hartmann-Hahn (HOHAHA)- type artifacts (25). A homospoil pulse was applied before each acquisition cycle. Spectra were acquired in the phase-sensitive mode with quadrature detection accomplished by the hypercom- o +30 method 304 increments with 24 scans E plex phase-cycling (26); a each were collected over a 5000-Hz width, zero-filled to E +20 spectral +o a 4K x 4K matrix, and transformed with Gaussian weighting in +10O both dimension. A spline-fitted baseline correction was applied in the Fl dimension after the Fourier transform. 0 CD spectroscopy was carried out in 1-mm strain-free quartz cells at 25°C in pure water using an Aviv Associates (Lake- x -10 wood, NJ) model 62 DS CD spectrometer. Data were taken with a 0.5-nm step size, and 4-s average time; the results were -20 averaged over 6 scans. Ellipticity is reported as mean residue ellipticity (n = 12). The concentration was determined by 190 210 230 250 270 amino acid analysis and by quantitative ninhydrin analysis A (nm) using calibration curves prepared from Ala, Lys, and NH3. The CD spectrum of Fig. 2 is typical for members of the Ac-Hel1- FIG. 2. The CD spectrum of Ac-Hel1-Alas-Lys-Ala6-NH2 in water Ala5-Lys-Alan-NH2 series in water. It is invariant to dilution at 25°C. Ellipticity values are uncorrected for the ellipticity attribut- and consistent with the able to the template, for the 28% of the peptide in the random coil cs- (27, 28) presence of helical te-states of and ts-states, and for the 36% nonhelical character within the te-state, a 12-peptide, at the abundance calculated from the measured calculated from the s values. The uncorrected molar ellipticity per t/c ratio. residue at 222 nm of -22,000 is in satisfactory agreement with values Using the general procedure described elsewhere (13), from reported for peptides of comparable size (27, 28). Ten-fold dilution a set of tic values for Ac-Hell-Alan-NH2, n = 0-6 and for had no effect on the CD spectrum. Downloaded by guest on September 29, 2021 Chemistry: Groebke et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4027

I A 5

5 B 4 '/I 10 4 9 2 8 3.- 1 7

I I I I I I I I 3 6 9 12 3 4 5 6 Number of H bonds Lysine site FIG. 3. (A) Plot of t/c values measured in water at 25°C as a function of the number of amide NH functions in the peptide chain, including the C-terminal NH2. Data points are shown for members of the series Ac-Hell-Ala,-NH2 (n = N-l, open circles) and for the series Ac-Hel1-Ala5-Lys-Alam-NH2 (m = N-7, filled circles). The lack of curvature within each series implies a value for SAla near 1.0 (11, 13). The graph includes t/c values for site variants Ac-Hell-Alas-Ala-NH2 and Ac-Hel1-Alas-Lys-NH2 (arrow, N = 7). Comparison of their t/c values shows that at this site, SLys > SAla. The steeper slope for filled circle data from lysine-containing peptides reflects the large value of SLys, consistent with the data analysis discussed below. The dotted line of the graph is calculated for SAla = 1.36. For the peptide pair with N = 7 (arrow), the least-squares fit for this value would imply that t/C(Ac-Hell-Aa5-Ala-NH2) > t/C(Ac-He1l-AIa5-Lys-NH2), contrary to experiment. The solid line of the graph is calculated for the iteratively derived optimal SAla = 1.025; here the fit for the pair at N = 7 is consistent with experiment. The lines of the graph are calculated from least-squares-derived constants A, B, and C and the block of 13 equations [where s = SAIa; (t/c)o = A]; C = SLysB(SA1a5): (t/c)N = A + B(sN-1)/(s-1), (N = 2-7, open circles), and (t/c)N = A + B(s6 - 1)/(s - 1) + C(s(N-6) - 1)/(s - 1)), (N = 7-13, filled circles) (10, 11, 13). For SAla = 1.025, A = 0.83 + 0.08, B = 0.153 + 0.016, C = 0.48 + 0.013, and SLys-6 = 2.7 + 0.4. The SAla values for the second NHs of RCO-Ala-Ala-OH and RCO-Ala-NH2 are equal within experimental error. (B) Plot of tic value as a function of lysine position i, measured in water at 25°C. Lysine was substituted sequentially for a single alanine residue at site i in Ac-Hel1-Alan-NH2 where n = 7 (open circles), 8 (0), 9 (o), and 10 (-). For each series, tic and therefore the helical stability increases significantly as the lysine residue is moved from site 3 to site 5. Given SAla < SLys, a constant SLys would result in a decrease of t/c with increasing i, which is seen for sites 5 and 6. The observed data are consistent with an i-dependent SLys that converges to a constant value with sufficient separation from the peptide-template junction. The lines correspond to t/c values calculated from a 41-point least-squares analysis that treats SLys as dependent on the peptide site i, counted from the template junction. Assumptions of a dependence of SLys on i counted from the C terminus or of a position-independent SLys give substantially poorer least-squares fits. Ac-Hell-Ala5-Lys-Alan-NH2, n = 0-6 in water, SAla values such as those for the short Ac-Hell-Alan-NH2 series, are were obtained by an iterative least-squares analysis. In the first omitted from the analysis. iteration, SAla = s is assigned an arbitrary initial value, and a Because only one lysine residue is present in each, the best fit for A, B, and C is calculated from Eq. 1. These are used peptides of the database have very high Ala-Lys ratios, and the to calculate 2 (tiCexperimental - t/Ccalc)2, which is dependent on overall least-squares goodness of fit is strongly determined by the choice of SAla. In subsequent iterations, SAla is varied to SAla, which is defined relatively precisely; SLys is less well minimize this error term. Least-squares analyses were done by defined. Within an acceptable range of least-squares error, a using the matrix equation (29): z = Inverse[(Transpose[x]) x)]. series of pairs of inversely correlated SLys and SAla values can [(Transpose[x])y), where y is the t/c data vector, which includes be chosen; a choice of a small SAla necessitates a large SLys and versa. more results from the n = 0 data point of t/c = 0.79; x = {{1,0},{1, (s6 - 1)/ vice A precise assignment of SLys (s - 1), 1}, {(s6 1)/(s - 1), (s2 1)/(s - 1)},..., {(s6 - 1)/ studies of alanine-rich peptide conjugates containing several residues at sites the chain (s - 1),(s7 - 1)/(s - 1))}};z = {A, B, C}, in which A, B, and C lysine widely separated along are defined as in Eq. 1 (10, 12, 13). (K.Y.T., P.R., and D.S.K., unpublished data). t/c = A + B[(s6 1)/(s 1)] + C[(sn+ 1)/(s - 1)], [1] RESULTS AND DISCUSSION where SLys = C/(BS5). Plots of tic vs. n for the series Ac-Hell-Ala5-Lys-Alan-NH2 or For the 41-member tic data set measured for Ac-Hel1-Alan- for the series Ac-Hell-Alan-NH2 show barely detectable pos- Lys-Alam-NH2, assignments of SAla and position-dependent itive curvature (Fig. 3A), and least-squares analysis for the values for SLys were obtained in an analogous manner. In this combined t/c data set gives SAla = 1.025. A 41-member data analysis we assumed that the value for SLys depends on the set including Ac-Hell-Alan-Lys-Alam-NH2, derivatives with n separation of the lysine site from the template-peptide junc- = 2-5 and m of 1-7, yields SAla = 1.01. The SAla = 1.07 reported tion. Alternative hypotheses were tested in which SLys depends by Scheraga and colleagues (30) from oligopeptide melting on separation from the C terminus or on the overall peptide curves is in near agreement with our SAla values. The analysis length, but these were found to give substantially larger of (Ala4-Lys)n by Baldwin and coworkers (2, 31-33) assigns a least-squares errors after the iterative optimization. An insig- passive role to lysine and yields much larger values for sAla that nificant variation in SAla is observed if selected blocks of data, do not agree with our results. Downloaded by guest on September 29, 2021 4028 Chemistry: Groebke et al. Proc. Natl. Acad. Sci. USA 93 (1996) We probed the helix-enhancing role of the length and charge residue and the peptide C terminus is shown in Fig. 3B. The of the lysine side chain by measuring tic for analogs Ac-Hell- data are consistent with a significant increase in SLys with Ala5-(NH-CH-X-CO)-NH2 that differ in structure only with increased distance from the N terminus. Study of alanine-rich respect to side-chain X. For these, an increase in tic implies a Ac-Hell conjugates containing two or more lysine residues larger sx. For X = -(CH2)n-H the tic values are 2.05 (n = 1, permits precise assignment of SLys as a function of position, as alanine), 2.08 (n = 2), 2.15 (n = 3), 2.25 (n = 4, norleucine); will be reported elsewhere (K.Y.T., P.R., and D.S.K., unpub- sx is seen to increase with additional hydrophobic surface area. lished data). For X = (CH2)n-NH3, the t/c values are 2.45 (n = 1), 2.3 (n Fig. 1B depicts one plausible structural model for the = 2), 2.2 (n = 3, ornithine), and 2.48 (n = 4, lysine). The interaction of the Lys e-NH3 with the helix core. For this substantial increase in tic value seen for replacement of -H by compact conformation, protons of all CH2 groups of the lysine -NH3 at an alanine 3-site is consistent with helix stabilization side chain approach the a-CH of the alanine at site (i - 3), and arising from interaction ofthe charge with the helix dipole. The protons of Lys NH3 are in contact with the (i - 4) carbonyl effect diminishes for the next two homologues, reflecting a oxygen. As seen in Fig. 4, NOEs observed for 'H NMR spectra larger separation between the cation and the core, with more of selectively deuterated analogs of Ac-Hel1-Ala7-Lys-NH2 efficient electrostatic screening by water. However, the incre- validate this model. Medium intensity interactions are seen ment for Nle -> Lys reverses this trend, plausibly reflecting an between the (i - 3) alanine a-CH and /3-, y-, and 8-CH2 unusually efficient charge-dipole interaction between the protons of the lysine, as well as a weak interaction with the Lys-NH3 ion and a carbonyl group of the helix core. Unlike the e-CH2. Ac-Hel1-Ala4-Lys-Ala3-NH2 shows medium intensity hydrophobic effect and ir hydrogen bonding, a charge-dipole interactions between the (i - 3) alanine a-CH and (3- and stabilization must show a strong position dependence. The 8-CH2 protons of the lysine, but lacks detectable y and E effect on tic of changing the separation between the lysine interactions.

A 1 23 4 DDm -

1.4 - 0 <(- 7 4- 8

- 1.8 4- a' 4-

2.2 -

2.6-

3.0 Io -- E C

.....~~~~~~~~~~~~~~~~~I!! 4.3 4.2 4.1 ppm B FIG. 4. 1H NMR spectra of Ac-Heli-4444344-(K-di)-NH2, a selectively deuterated analog of Ac-Hell-Ala7-Lys-NH2 (abbreviations: 4, L-Ala-2,3,3,3-d4; 3, L-Ala-3,3,3-d3; K-dl, L-Lys-2-dl). (A) Expansion of one-dimensional spectrum. (B) Two-dimensional rotating-frame Overhauser effect spectroscopy spectrum. The selected region shows crosspeaks between the 3, y, 8, and e protons of the lysine side chain and the undeuterated a position at site (i - 3). (C) The observed NOE crosspeaks are consistent with a peptide conformation in which the lysine side chain packs against the helix barrel. It is noteworthy that only the helical peptide fraction associated with a t-state template moiety (see Fig. 1C) shows NOE crosspeaks between the lysine methylene signals and the signal for the a-proton at site (i - 3) (labeled 3 in A). For the random coil fraction of the peptide associated with a c-state template moiety, the corresponding NOE crosspeaks for the a proton at site (i - 3) (labeled 2 in A) are not observed. This result shows that a packed conformation of the lysine side chain resulting in NOE crosspeaks at the (i - 3) site is significantly populated only within a helical arrangement of the peptide backbone. A rotating-frame Overhauser effect spectroscopy spectrum (data not shown) for Ac-Hell-4344-(K-di)-444-NH2, a deuterated analog of Ac-Hell-Ala4-Lys-Ala3-NH2, contains crosspeaks between the 3 and 8 methylene signals of the central lysine residue and the signal for the a proton at site (i - 3), again indicating a compact conformation, but with a slightly different arrangement of the polymethylene chain with respect to the torsional angles. No rotating-frame Overhauser effect interactions between the lysine side chain and either the a-proton at site (i - 4) or the methyl groups at site (i - 3) or (i - 4), respectively, were observed in appropriately deuterated derivatives of Ac-Heli-Ala7-Lys-NH2 and Ac-Hell-Ala4-Lys-Ala3-NH2 (spectra not shown). Downloaded by guest on September 29, 2021 Chemistry: Groebke et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4029 The large change in tic observed for the structural change 2. Rohl, C. A., Scholtz, J. M., York, E. J., Stewart, J. M. & Baldwin, (CH2)4-H --- (CH2)4-NH3 is not seen for (CH2)3-H (CH2)3- R. L. (1992) Biochemistry 31, 1263-1269. NH'3. This dependence of energetics on the length of the 3. Merutka, G. & Stellwagen, E. (1990) Biochemistry 29, 894-898. methylene linkage between ammonium ion and peptide back- 4. Merutka, G., Shalongo, W. & Stellwagen, E. (1991) Biochemistry bone implies a unique stabilization mechanism for lysine, 30, 4245-4248. consistent with significant populations of packed conforma- 5. Todd, A. P. & Millhauser, G. L. (1991) Biochemistry 30, 5515- tions such as that of Fig. lB. The presence of these is confirmed 5523. by the NOE analyses. Our estimates of SLys derived from the 6. Fiori, W. R., Miick, S. M. & Millhauser, G. L. (1993) Biochem- full of 41 derivatives fall in the of istry 32, 11957-11962. least-squares analysis range 7. Chakrabartty, A., Kortemme, T. & Baldwin, R. L. (1994) Protein 1.3-3.5, with a significant positive correlation with distance of Sci. 3, 843-852. lysine from the N terminus, consistent with the scan of Fig. 3B. 8. Scholtz, J. M., York, E. J., Stewart, J. M. & Baldwin, R. L. (1991) These large SLys values are exceptional and noteworthy but are J. Am. Chem. Soc. 113, 5102-5104. consistent with the helicity reported for certain short lysine- 9. Vila, J., Williams, R. L., Grant, J. A., Wojcik, J. & Scheraga, containing peptides. They are in complete accord with the H. A. (1992) Proc. Natl. Acad. Sci. USA 89, 7821-7825. modeling predictions of Vila et al. (9), as well as with the recent 10. Kemp, D. S., Curran, T. P., Boyd, J. G. & Allen, T. (1991)J. Org. suggestion of Chakrabartty et al. (7) concerning the helix- Chem. 56, 6683-6697. stabilizing role of side-chain interactions. 11. Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (Lon- Two pertinent questions concern the generality of our don) 352, 451-454. findings and the variance between our conclusions and those 12. Kemp, D. S., Allen, T. J. & Oslick, S. L. (1995) J. Am. Chem. Soc. of Chakrabartty et al. (7). We defer a full discussion ofvariance 117, 6641-6657. to a report of the helicities of peptide conjugates containing 13. Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. more than one lysine residue. Here we note that for a given 118, in press. data set, allowable choices for SAla and SLys are expected to 14. Poland, D. & Scheraga, H. A. (1970) Theory of the Helix-Coil show a strong statistical inverse correlation. In most cases SAla Transitions in Biopolymers (Academic, New York). and SLys cannot be assigned uniquely, but are definable as a 15. Nemethy, G. & Scheraga, H.A. (1962) J. Phys. Chem. 66, series of fixed in the allowed overall 1773-1789. pairs precision by error 16. Nemethy, G., Steinberg, I. Z. & Scheraga, H. A. (1963) Biopoly- and by the covariance properties of the data. For the 56 mers 1, 43-63. lysine-containing peptides studied by Chakrabartty et al. (7), 17. Fairman, R., Schoemaker, K. H., York, E. J., Stewart, J. M. & the Ala/Lys ratios fall in a narrow range close to 2.9. For the Baldwin, R. L. (1989) Proteins Struct. Funct. Genet. 5, 1-7. lysine-containing conjugates of the present study, the Ala/Lys 18. Perutz, M. F. & Fermi, G. (1988) Proteins Struct. Funct. Genet. ratios vary from 5 to 11. Other factors being equal, high values 4, 294-295. and a large range for Ala/Lys should permit SAla to be defined 19. Hol, W. G. J., van Duijnen, P. T. & Berendsen, H. J. C. (1978) with greater precision. Nature (London) 273, 443-445. We observe these correlations for lysine in an alanine 20. Hol, W. G. J., Halie, L. M. & Sander, C. (1981) Nature (London) matrix, free of other amino acids. Do they hold more gener- 294, 532-534. ally? Modeling (Fig. 1) suggests that optimal lysine stabiliza- 21. Lotun, N., Yaron, A. & Berger, A. (1966)Biopolymers 4,365-368. tion requires an interaction of the lysine e-NH3 with an (i - 22. Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, C. D. & 4) backbone carbonyl, which is perturbed by bulky side chains Jeanloz, R. W. (1984) J. Am. Chem. Soc. 106, 811-813. at sites (i - 3) and (i - 4). Maximal lysine stabilization may 23. Bax, A. & Davis, D. G. (1985) J. Magn. Reson. 63, 207-213. well result only if alanine occupies these sites, implying that a 24. Kessler, H., Griesinger, C., Kerssebaum, R., Wagner, K. & Ernst, combination of with alanine at sites - and - R. R. (1987) J. Am. Chem. Soc. 109, 607-609. lysine (i 3) (i 4) 25. Farmer, B. T., II, & Brown, L. R. (1987) J. Magn. Reson. 72, generates that stabilization. For alanine-rich peptides, others 197-202. have noted the helix-stabilizing effect of amino acids bearing 26. States, D. J., Haberkorn, R. A. & Ruben, D. J. (1982) J. Magn. straight chains terminating in charged or polar groups, with a Reson. 48, 286-292. rank order: Arg > Lys, Glu > Gln, Orn (1, 4, 8, 34). For these 27. Dyson, H.J., Rance, M., Houghten, R. A., Wright, P. E. & amino acids, we conjecture that helical stabilization results Lerner, R. L. (1988) J. Mol. Biol. 201, 201-217. primarily from interactions of the side chain with the helix 28. Lehrman, S. R., Tuls, J. L. & Lund, M. (1990) Biochemistry 29, barrel, implying sensitivity of s values to amino acid substitu- 5991-5596. tion at the (i - 3) and (i - 4) sites. For 10 cases that have been 29. Hamilton, W. C. (1994) Statistics in Physical Science (Ronald studied, substitution of arginine for lysine in our conjugates Press, New York), pp. 124-132. increases t/c substantially. 30. Wojcik, J., Altmann, K.-H. & Scheraga, H. A. (1990) Biopolymers 30, 121-134. This work was supported by the National Science Foundation 31. Chakrabartty, A., Schellmann, J. A. & Baldwin, R. L. (1991) (9121702-CHE), the National Institutes of Health (GM 13453), Pfizer Nature (London) 351, 586-588. Research, the Swiss National Science Foundation (fellowships to K.G. 32. Scholtz, J. M., Qian, H., York, E. J., Stewart, J. M. & Baldwin, and P.R.) and the Roche Research Foundation (fellowship to P.R.). R. L. (1991) Biopolymers 31, 1463-1470. 33. Chakrabartty, A., Kortemme, T., Padmanabhan, S. & Baldwin, 1. Marqusee, S., Robbins, V. H. & Baldwin, R. L. (1989) Proc. Natl. R. L. (1993) Biochemistry 32, 5560-5565. Acad. Sci USA 86, 5286-5290. 34. Merutka, G. & Stellwagen, E. (1991) Biochemistry 30, 1591-1594. Downloaded by guest on September 29, 2021