A critical assessment of putative gatekeeper interactions in the villin headpiece helical subdomain.

The helical subdomain of the villin headpiece (HP36) is one of the smallest naturally occurring proteins that folds cooperatively. Its small size, rapid folding, and simple three-helix topology have made it an extraordinary popular model system for computational, theoretical, and experimental studies of protein folding. Aromatic-proline interactions involving Trp64 and Pro62 have been proposed to play a critical role in specifying the subdomain fold by acting as gatekeeper residues. Note that the numbering corresponds to full-length headpiece. Mutation of Pro62 has been shown to lead to a protein that does not fold, but this may arise for two different reasons: The residue may make interactions that are critical for the specificity of the fold or the mutation may simply destabilize the domain. In the first case, the protein cannot fold, while in the second, the small fraction of molecules that do fold adopt the correct structure. The modest stability of the wild type prevents a critical analysis of these interactions because even moderately destabilizing mutations lead to a very small folded state population. Using a hyperstable variant of HP36, denoted DM HP36, as our new wild type, we characterized a set of mutants designed to assess the role of the putative gatekeeper interactions. Four single mutants, DM Pro62Ala, DM Trp64Leu, DM Trp64Lys, and DM Trp64Ala, and a double mutant, DM Pro62Ala Trp64Leu, were prepared. All mutants are less stable than DM HP36, but all are well folded as judged by CD and (1)H NMR. All of the mutants display sigmoidal thermal unfolding and urea-induced unfolding curves. Double-mutant cycle analysis shows that the interactions between Pro62 and Trp64 are weak but favorable. Interactions involving Pro62 and proline-aromatic interactions are, thus, not required for specifying the subdomain fold. The implications for the design and thermodynamics of miniature proteins are discussed.

A critical assessment of the role of helical intermediates in amyloid formation by natively unfolded proteins and polypeptides.

Amyloidogenic proteins and polypeptides can be divided into two structural classes, namely those which are flexible and are intrinsically disordered in their unaggregated state and those which form a compact globular structure with a well-defined tertiary fold in their normally soluble state. This review article is focused on amyloid formation by natively disordered polypeptides. Important examples of this class include islet amyloid polypeptide (IAPP, amylin), pro-IAPP processing intermediates, alpha-synuclein, the Abeta peptide, atrial natriuretic factor, calcitonin, pro-calcitonin, the medin polypeptide, as well as a range of de novo designed peptides. Amyloid formation is a complex process consisting of a lag phase during which no detectable fibril material is formed, followed by a rapid growth phase that leads to amyloid fibrils. A critical analysis of the literature suggests that a subset of intrinsically disordered polypeptides populate a helical intermediate during the lag phase. In this scenario, early formation of multimeric species is promoted by helix-helix association involving one region of the polypeptide chain which leads to a high effective concentration of an amyloidogenic sequence located in a different region of the chain. Helical intermediates appear to be particularly important in membrane-catalyzed amyloid formation and have been implicated in glycosaminoglycan mediated amyloid formation as well. There is suggestive evidence that targeting helix-helix interactions can be a viable strategy to inhibit amyloid formation. The characterization of transient helical intermediates is challenging, but new methods are being developed that offer the prospect of providing residue-specific information in real time.

A simple and economical method for the production of 13C,18O-labeled Fmoc-amino acids with high levels of enrichment: applications to isotope-edited IR studies of proteins.

Isotope-edited IR of proteins has generated considerable interest. Double labeling with 13C and 18O with high levels of isotopic enrichment is required for residue-specific resolution. Current methods for the preparation of doubly labeled amino acids give modest 18O enrichment, limiting the utility of the approach. We report a simple and economical method for preparing 13C,18O-doubly labeled N-(9-fluorenylmethoxycarbonyl)amino acids with high levels of enrichment for residues that do not require acid-labile side-chain protecting groups.

Beyond the decoupling approximation in the model free approach for the interpretation of NMR relaxation of macromolecules in solution.

The model free (MF) approach allows straightforward extraction of generalized order parameters and correlation times for internal and overall bond vector reorientational fluctuations from NMR spin relaxation measurements for macromolecules in solution. The drawback of this approach is the use of a decoupling approximation that neglects correlations between internal and overall molecular motions. These correlations are significant when fluctuation amplitudes are less than the size of the "cage" that restricts the bond vector. In this regime, motion of the bond vector is only indirectly affected by molecule fluctuations through the orienting potential for the local director axis. By separating the regimes of large and small amplitude molecular fluctuations, an expression for the spectral density function is derived that does not invoke the decoupling approximation, but that relies on the same macroscopic level of description as the original MF approach. The parameters of the spectral density function provide insight into the effects on the MF parameters that result from violation of the decoupling approximation.