Design of stable alpha-helical arrays from an idealized TPR motif

The tetratricopeptide repeat (TPR) is a 34-amino acid alpha-helical motif that occurs in over 300 different proteins. In the different proteins, three to sixteen or more TPR motifs occur in tandem arrays and function to mediate protein-protein interactions. The binding specificity of each TPR protein is different, although the underlying structural motif is the same. Here we describe a statistical approach to the design of an idealized TPR motif. We present the high-resolution X-ray crystal structures (to 1.55 and 1.6 A) of designed TPR proteins and describe their solution properties and stability. A detailed analysis of these structures provides an understanding of the TPR motif, how it is repeated to give helical arrays with different superhelical twists, and how a very stable framework may be constructed for future functional designs.     

Modulating repeat protein stability: the effect of individual helix stability on the collective behavior of the ensemble

Repeat proteins are tandem arrays of a small structural motif, in which tertiary structure is stabilized by interactions within a repeat and between neighboring repeats. Several studies have shown that this modular structure is manifest in modular thermodynamic properties. Specifically, the global stability of a repeat protein can be described by simple linear models, considering only two parameters: the stability of the individual repeated units (H) and the coupling interaction between the units (J). If the repeat units are identical, single values of H and J, together with the number of repeated units, is sufficient to completely describe the thermodynamic behavior of any protein within a series. In this work, we demonstrate how the global stability of a repeat protein can be changed, in a predictable fashion, by modifying only the H parameter. Taking a previously characterized series of consensus tetratricopeptide repeats (TPR) (CTPRa) proteins, we introduced mutations into the basic repeating unit, such that the stability of the individual repeat unit was increased, but its interaction with neighboring units was unchanged. In other words, we increased H but kept J constant. We demonstrated that the denaturation curves for a series of such repeat proteins can be fit and additional curves can be predicted by the one-dimensional Ising model in which only H has changed from the original fit for the CTPRa series. Our results show that we can significantly increase the stability of a repeat protein by rationally increasing the stability of the units (H), whereas the interaction between repeats (J) remains unchanged.     

Tracking the unfolding pathway of a multirepeat protein via tryptophan scanning: evidence of localized instability in the mitochondrial import receptor Tom70

The tetratricopeptide repeat (TPR) is a degenerate 34-amino acid repeating motif that forms a repeating helix-turn-helix structure and is a well characterized mediator of protein-protein interactions. Recently, a biophysical investigation on one naturally occurring TPR protein, Tom70, found that the mitochondrial receptor displayed an unusual three-state unfolding pathway, distinct from the two-state model usually displayed by TPR proteins. To investigate this unusual behavior, we undertook a tryptophan-scanning analysis of Tom70, where both native and engineered tryptophan residues are used as fluorescent reporters to monitor the range of local and global unfolding events that comprise the unfolding pathway of Tom70. Specifically, seven Tom70 variants were constructed, each with a single tryptophan residue in each of the seven TPR repeats of Tom70. By combining equilibrium and kinetic fluorescent unfolding assays, with circular dichroism experiments, our study reveals that the unusual folding pathway of Tom70 is a consequence of the unfolding of two separate, autonomous TPR arrays, with the less stable region appearing to account for the low structural stability of Tom70.     

Mapping the energy landscape of repeat proteins using NMR-detected hydrogen exchange

Repeat proteins contain tandem arrays of a simple structural motif. In contrast to globular proteins, repeat proteins are stabilized only by interactions between residues that are relatively close together in the sequence, with no ”long-range” interactions. Our work focuses on the tetratricopeptide repeat (TPR), a 34 amino acid helix-turn-helix motif found in tandem arrays in many natural proteins. Earlier, we reported the design and characterization of a series of consensus TPR (CTPR) proteins, which are built as arrays of multiple tandem copies of a 34 amino acid consensus sequence. Here, we present the results of extensive hydrogen exchange (HX) studies of the folding-unfolding behavior of two CTPR proteins (CTPR2 and CTPR3). We used HX to detect and characterize partially folded species that are populated at low frequency in the nominally folded state. We show that for both proteins the equilibrium folding-unfolding transition is non-two-state, but sequential, with the outermost helices showing a significantly higher probability than inner helices of being unfolded. We show that the experimentally observed unfolding behavior is consistent with the predictions of a simple Ising model, in which individual helices are treated as ”spin-equivalents”. The results that we present have general implications for our understanding of the thermodynamic properties of repeat proteins.     

Calorimetric study of a series of designed repeat proteins: modular structure and modular folding

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.     

Functional comparison of human and Drosophila Hop reveals novel role in steroid receptor maturation

Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.     

The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold

There are several different families of repeat proteins. In each, a distinct structural motif is repeated in tandem to generate an elongated structure. The nonglobular, extended structures that result are particularly well suited to present a large surface area and to function as interaction domains. Many repeat proteins have been demonstrated experimentally to fold and function as independent domains. In tetratricopeptide (TPR) repeats, the repeat unit is a helix-turn-helix motif. The majority of TPR motifs occur as three to over 12 tandem repeats in different proteins. The majority of TPR structures in the Protein Data Bank are of isolated domains. Here we present the high-resolution structure of NlpI, the first structure of a complete TPR-containing protein. We show that in this instance the TPR motifs do not fold and function as an independent domain, but are fully integrated into the three-dimensional structure of a globular protein. The NlpI structure is also the first TPR structure from a prokaryote. It is of particular interest because it is a membrane-associated protein, and mutations in it alter septation and virulence.     

p62cdc23 of Saccharomyces cerevisiae: a nuclear tetratricopeptide repeat protein with two mutable domains.

CDC23 is required in Saccharomyces cerevisiae for cell cycle progression through the G2/M transition. The CDC23 gene product contains tandem, imperfect repeats, termed tetratricopeptide repeats, (TPR) units common to a protein family that includes several other nuclear division CDC genes. In this report we have used mutagenesis to probe the functional significance of the TPR units within CDC23. Analysis of truncated derivatives indicates that the TPR block of CDC23 is necessary for the function or stability of the polypeptide. In-frame deletion of a single TPR unit within the repeat block proved sufficient to inactivate CDC23 in vivo, though this allele could rescue the temperature-sensitive defect of a cdc23 point mutant by intragenic complementation. By both in vitro and in vivo mutagenesis techniques, 17 thermolabile cdc23 alleles were produced and examined. Fourteen alleles contained single amino acid changes that were found to cluster within two distinct mutable domains, one of which encompasses the most canonical TPR unit found in CDC23. In addition, we have characterized CDC23 as a 62-kDa protein (p62cdc23) that is localized to the yeast nucleus. Our mutagenesis results suggest that TPR blocks form an essential domain within members of the TPR family.     

New materials from proteins and peptides

In this review we highlight recent accomplishments in the design of materials from proteins and peptides. Examples include hydrogels made from aggregating designed β-hairpin peptides, whose physical properties respond to small changes in the amino acid composition of the peptide; materials that combine different segments of natural elastomeric proteins - such as elastin, resilin, silk fibroin whose bulk properties are dictated in unanticipated ways by their composition; and hydrogels formed by strings or arrays of protein modules, which are cross-linked by multivalent versions of their peptide ligands, and which may exhibit exquisite stimuli-responsive behavior. The suitability of the unique properties of such new materials for practical applications is also considered.     

Between-species analysis of short-repeat modules in cell wall and sex-related hydroxyproline-rich glycoproteins of Chlamydomonas

Protein diversification is commonly driven by single amino acid changes at random positions followed by selection, but, in some cases, the structure of the gene itself favors the occurrence of particular kinds of mutations. Genes encoding hydroxyproline-rich glycoproteins (HRGPs) in green organisms, key protein constituents of the cell wall, carry short-repeat modules that are posited to specify proline hydroxylation and/or glycosylation events. We show here, in a comparison of two closely related Chlamydomonas species-Chlamydomonas reinhardtii (CC-621) and Chlamydomonas incerta (CC-1870/3871)-that these modules are prone to misalignment and hence to both insertion/deletion and endoduplication events, and that the dynamics of the rearrangements are constrained by purifying selection on the repeat patterns themselves, considered either as helical or as longitudinal face modules. We suggest that such dynamics may contribute to evolutionary diversification in cell wall architecture and physiology. Two of the HRGP genes analyzed (SAG1 and SAD1) encode the mating-type plus and minus sexual agglutinins, displayed only by gametes, and we document that these have undergone far more extensive divergence than two HRGP genes (GP1 and VSP3) that encode cell wall components-an example of the rapid evolution that characterizes sex-related proteins in numerous lineages. Strikingly, the central regions of the agglutinins of both mating types have diverged completely, by selective endoduplication of repeated motifs, since the two species last shared a common ancestor, suggesting that these events may have participated in the speciation process.     

Comparison of the backbone dynamics of a natural and a consensus designed 3-TPR domain

The tetratricopeptide repeat (TPR) is a 34-amino acid helix-turn-helix motif that occurs in tandem arrays in numerous proteins. Here we compare the backbone dynamics of a natural 3-repeat TPR domain, from the protein UBP, with the behavior of a designed protein CTPR3, which consists of three identical consensus TPR units. Although the three tandem TPR repeats in both CTPR3 and UBP behave as a single unit, with no evidence of independent repeat motions, the data indicate that certain positions in UBP are significantly more flexible than are the corresponding positions in CTPR3. Most of the dynamical changes occur at or adjacent to positions that are involved in intra-repeat packing interactions. These observations lead us to suggest that the three-TPR domain of UBP does not incorporate optimized packing, compared to that seen in the idealized CTPR. The natural TPR domain is not only less stable overall than CTPR3, but also presents increased local flexibility at the positions where the sequences differs from the conserved consensus.     

Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module

Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.     

Elastin-like Peptide amphiphiles form nanofibers with tunable length

Peptide amphiphiles (PAs) self-assemble nanostructures with potential applications in drug delivery and tissue engineering. Some PAs share environmentally responsive behavior with their peptide components. Here we report a new type of PAs biologically inspired from human tropoelastin. Above a lower critical solution temperature (LCST), elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition. Similar to other PAs, elastin-like PAs (ELPAs) assemble micelles with fiber-like nanostructures. Similar to ELPs, ELPAs have inverse phase transition behavior. Here we demonstrate control over the ELPAs fiber length and cellular uptake. In addition, we observed that both peptide assembly and nanofiber phase separation are accompanied by a distinctive secondary structure attributed primarily to a type-1 β turn. We also demonstrate increased solubility of hydrophobic paclitaxel (PAX) in the presence of ELPAs. Due to their biodegradability, biocompatibility, and environmental responsiveness, elastin-inspired biopolymers are an emerging platform for drug and cell delivery; furthermore, the discovery of ELPAs may provide a new and useful approach to engineer these materials into stimuli-responsive gels and drug carriers.     

Molecular recognition via coupled folding and binding in a TPR domain

The majority of known tetratricopeptide repeat (TPR) domains consist of three copies of the helix-turn-helix TPR motif, together with a seventh C-terminal helix. TPR domains function as protein-protein recognition modules in intracellular signalling. This function is exemplified by the TPR domain of protein phosphatase 5 (PP5), which binds to the C terminus of the chaperone protein Hsp90. Here, we report NMR and CD spectroscopic studies that reveal that this domain is largely unfolded at physiological temperatures, and that interaction with an MEEVD pentapeptide derived from Hsp90 stabilises a folded structure. This complex, coupled folding-binding mechanism is characterised further by its observed enthalpy change on binding (determined by isothermal titration calorimetry), which displays a markedly non-linear relationship with temperature. A nested Gibbs-Helmholtz model is used in a novel combined analysis of the CD and ITC data to determine separately the thermodynamic contributions of the intrinsic folding and binding events to the overall coupled process. The analysis shows that, despite the expected large entropic opposition to the folding process, a nearly equal favourable folding enthalpy means the net effect of coupled folding on the observed affinity is small across a broad range of temperature. We hypothesise that a coupled folding-binding mechanism is common in this class of domains.     

Studies of the Inheritance of Human Ribosomal DNA Variants Detected in Two-Dimensional Separations of Genomic Restriction Fragments

We have investigated the variation in human ribosomal DNA repeat units as revealed in two-dimensional electrophoretic separations of genomic restriction fragments that were end-labeled at NotI cleavage sites. The transcribed portion of the ribosomal DNA results in ~20 labeled fragments visible on each gel as multicopy spots. We have mapped these spots to the sequences responsible for their appearance on the gels, based on their migration positions and direct sequencing of spots, and describe several previously unreported sources of variation. By studying mother/father/child families we gained information on how much of the between-repeats variation is due to differences between and within repeat arrays on homologous chromosomes. Two instances in which a child exhibited more copies of a particular fragment than were present in the parents are described and hypothesized to be due to events such as multiple unequal sister-chromatid exchanges or gene conversions.     

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.