Impact of strand length on the stability of parallel-β-sheet secondary structure.

The thousands of tertiary structural motifs known among natural proteins are built up from a handful of regular secondary structures, mostly a helices and b sheets, which are linked by b turns and larger loops. Sequence–stability relationships for a helices and for antiparallel b sheets have been widely explored using autonomously folding peptides, an experimental approach that eliminates the potentially confounding influence of a specific tertiary context. In contrast, a minimal parallel b sheet cannot readily be generated within a conventional peptide because the N terminus of one strand is necessarily distant from the C terminus of a neighboring strand. A continuous peptide design for a parallel b sheet would require a long interstrand loop, introducing significant tertiary packing effects or an entropic penalty for folding, either of which would compromise the analysis. Thus, novel strategies are required to elucidate the factors that govern parallel-b-sheet stability. Herein we use recently developed tools, including short nonpeptidic units for connecting peptide strands through their C termini, to explore a fundamental question: does parallel-b-sheet secondary structure become more stable as the strands grow longer? The a helix is well known to be stabilized by increasing length. a-Helix initiation (ordering of the first four residues, to enable formation of the first intrahelical H-bond) entails a large entropic cost, which can be offset by a small but favorable energetic gain from propagation (incorporation of individual residues into a preexisting helix). The longer the a helix, the larger the net thermodynamic benefit from propagation, if the residues have a favorable helix propensity. In contrast, antiparallel-bsheet secondary structure appears to be subject to an intrinsic limit on strand length. The relationship between strand length and parallel-b-sheet stability has not previously been addressed. This gap in experimentation may underlie the relatively limited theoretical treatment of b-sheet formation, especially for parallel b sheets, which stands in contrast to the extensive theoretical analysis of a-helical folding. Herein, we show that parallel b sheets becomes steadily more stable as the strands are lengthened, if the added residues have a high b-sheet propensity. Our experimental design is outlined in Figure 1a. A diamine segment containing d-proline is used to link two strand-forming peptide segments through their C termini. This linker promotes but does not strictly enforce bsheet interactions between attached strands in aqueous solution. The extent of b-sheet formation is monitored by NMR spectroscopy by measuring a-proton chemical shifts (dCaH) at “indicator” residues (black dots) within the core segment (black) as the strands are extended incrementally (blue). The indicator residues are insulated from the extension segments by an intervening residue; therefore, changes in dCaH value induced by varying extension length should reflect a global change in b-sheet population. Our initial extension sequences contain valine (Val, V) and threonine (Thr, T), residues that have a high intrinsic bsheet propensity because of branching in the side chain. For the extensions shown in Figure 1a, each added Thr residue should form backbone H-bonds to the opposite strand, while each new Val should present its backbone Hbonding groups (C=O and N H) to the solvent. Members of this series are designated P(T!V)n, where n is the number of residues in each extension segment; the peptide is P when n= 0. Our design includes Coulombic attractions at the N termini of the strand segments (red), which are intended to minimize fraying of the open end of the parallel hairpin. Sedimentation equilibrium analytical ultracentrifugation, variable concentration NMR spectroscopy, and diffusion oriented spectroscopy (DOSY) NMR studies suggest that P and the other peptides discussed do not aggregate under the conditions used to obtain the NMR data (see Figures S8–S12 in the Supporting Information). Two-dimensional NMR analysis of parent peptide P indicates that the intended parallel-b-sheet conformation is formed in aqueous solution. Multiple NOE signals are observed between side chains that are distant from one another in terms of sequence, but that are expected to lie near one another in the parallel b sheet (see Figure S6 in the Supporting Information). No NOE signals inconsistent with the expected parallel-b-sheet conformation were observed. A set of ten low-energy structures for P (backbone only) derived from NOE-restrained molecular dynamics simulations is shown in Figure 2a. The structures vary significantly from one another, but the presence of a consistent hairpin fold is evident from the overlay. This image reflects a feature that is probably common to many peptides and small [*] Dr. F. Freire, A. M. Almeida, Dr. J. D. Fisk, J. D. Steinkruger, Prof. S. H. Gellman Department of Chemistry, University of Wisconsin–Madison 1101 University Av., Madison, WI 53726 (USA) E-mail: gellman@chem.wisc.edu [] These authors contributed equally to this work.