Surface-bound polypeptides and proteins are increasingly used to functionalize inorganic interfaces such as electrodes, but their structural characterization is exceedingly difficult with standard technologies. In this paper, we report the first two-dimensional sum-frequency generation (2D SFG) spectra of a peptide monolayer, which is collected by adding a mid-IR pulse shaper to a standard femtosecond SFG spectrometer. On a gold surface, standard FTIR spectroscopy is inconclusive about the peptide structure because of solvation-induced frequency shifts, but the 2D lineshapes, anharmonic shifts, and lifetimes obtained from 2D SFG reveal that the peptide is largely α-helical and upright. Random coil residues are also observed, which do not themselves appear in SFG spectra due to their isotropic structural distribution, but which still absorb infrared light and so can be detected by cross-peaks in 2D SFG spectra. We discuss these results in the context of peptide design. Because of the similar way in which the spectra are collected, these 2D SFG spectra can be directly compared to 2D IR spectra, thereby enabling structural interpretations of surface-bound peptides and biomolecules based on the well-studied structure/2D IR spectra relationships established from soluble proteins.
A new strategy for rapid evaluation of sequence-stability relationships in the parallel coiled-coil motif is described. The experimental design relies upon thiol-thioester exchange equilibria, an approach that is particularly well suited to examination of heterodimeric systems. Our model system has been benchmarked by demonstrating that it can quantitatively reproduce previously reported trends in interhelical a-a' side chain pairing preferences at the coiled-coil interface. This new tool has been used to explore the role of Coulombic interactions between a core position on one helix and a flanking position on the other helix (a-g'). This type of interhelical contact has received relatively little attention to date. Our results indicate that such interactions can influence coiled-coil partner preferences.Proteins collectively display a broad array of tertiary and quaternary structures, with many different modes of packing between neighboring secondary structure elements. Among the possibilities, the α-helical coiled coil is unusual in that it is both common and regular. 1 In the simplest case, two α-helices associate side-by-side, wrapping around one another with a slight left-handed superhelical twist. A characteristic "knobs-into-holes" interdigitation of sidechains is observed at the helix-helix interface, whether the helices are parallel or antiparallel. 2 The relative simplicity of this architecture has led to extensive exploration of sequencestability relationships, 3 motivated by the prospects of predicting coiled-coil structure from sequence information alone, refining computational tools, and using coiled coils as building blocks in rational protein design and synthetic biology. 4 Although some principles that govern coiled-coil stability have been elucidated, our understanding remains incomplete.Here we introduce a heterodimeric parallel coiled-coil model system designed to provide new insights on the origins of stability and helix-pairing preferences. Our system employs relatively short peptide segments (20 or 21 residues), which facilitates broad exploration of sequence variations. Parallel, two-helix assembly is promoted by a thioester linkage between the Cterminus of one segment and the side-chain of a C-terminal Cys residue on the other; this design enables us to monitor coiled-coil stability under native conditions via thiol-thioester exchange equilibration. 5 Our experimental design (Figure 1) is based on well-known characteristics of sequences that form coiled coils. The segments intended to adopt α-helical conformations feature a heptad sequence repeat pattern (abcdefg), in which side-chains at a and d dominate the helix-helix contacts. Two-helix stoichiometry (rather than alternate three-or four-helix assemblies) is directed by placing Leu at the d sites, Ile at the N-terminal a positions, and Asn at the a sites closest to the covalent connection.6 ,7 The remaining (central) a positions of each segment (designated X and Ψ) are "guest" sites for substitutions that allow us to probe ...
Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called ‘knobs-into-holes’ (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exhibit significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral at a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pair-wise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.
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