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Template-Nucleated Alanine-Lysine Helices Are Stabilized by Position-Dependent Interactions Between the Lysine Side

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Template-Nucleated Alanine-Lysine Helices Are Stabilized by Position-Dependent Interactions Between the Lysine Side 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.
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