Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein's superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.
Abstract Solvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D 2 O compared with H 2 O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H 2 O and D 2 O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate‐determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNature and Consequences of GroEL-Protein InteractionsLaura S. Itzhaki, Daniel E. Otzen, and Alan R. FershtCite this: Biochemistry 1995, 34, 44, 14581–14587Publication Date (Print):November 1, 1995Publication History Published online1 May 2002Published inissue 1 November 1995https://pubs.acs.org/doi/10.1021/bi00044a037https://doi.org/10.1021/bi00044a037research-articleACS PublicationsRequest reuse permissionsArticle Views149Altmetric-Citations64LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
Repeat proteins are composed of tandem arrays of 30- to 40-residue structural motifs and are characterized by short-range interactions between residues close in sequence. Here we have investigated the equilibrium unfolding of D34, a 426-residue fragment of ankyrinR that comprises 12 ankyrin repeats. We show that D34 unfolds via an intermediate in which the C-terminal half of the protein is structured and the N-terminal half is unstructured. Surprisingly, however, we find that we change the unfolding process when we attempt to probe it. Single-site, moderately destabilizing mutations at the C terminus result in different intermediates dominating. The closer to the C terminus the mutation, the fewer repeats are structured in the intermediate; thus, structure in the intermediate frays from the site of the mutation. This behavior contrasts with the robust unfolding of globular proteins in which mutations can destabilize an intermediate but do not cause a different intermediate to be populated. We suggest that, for large repeat arrays, the energy landscape is very rough, with many different low-energy species containing varying numbers of folded modules so the species that dominates can be altered easily by single, conservative mutations. The multiplicity of partly folded states populated in the equilibrium unfolding of D34 is also mirrored by the kinetic folding mechanism of ankyrin-repeat proteins in which we have observed that parallel pathways are accessible from different initiation sites in the structure.
The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats. However, close examination of the mutant chevron plots reveals that simple models are insufficient to describe all of the data, and double mutant analysis subsequently confirms that there are parallel folding pathways. Destabilizing mutations in the C-terminal repeats reduce flux through the wild-type pathway, making a new route accessible in which folding is initiated at the N-terminal repeats. Thus, the folding mechanism of the repeat protein is poised on a fulcrum: When one end of the molecule is perturbed, the balance shifts between the different nucleation sites. The vast majority of studies on small globular proteins indicate a single, well defined route between the denatured and native states. By contrast, the potential to initiate folding at more than one site may be a general feature of repeat proteins arising from the symmetry inherent in their structures. We show that this simple architecture makes it straightforward to direct the folding pathway of a repeat protein by design.
Stapled peptides have great potential as modulators of protein–protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure–activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.
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