Ewan R. G. Main

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.

Protein folding : Defining a "standard" set of experimental conditions and a preliminary kinetic data set of two-state proteins

Recent years have seen the publication of both empirical and theoretical relationships predicting the rates with which proteins fold. Our ability to test and refine these relationships has been limited, however, by a variety of difficulties associated with the comparison of folding and unfolding rates, thermodynamics, and structure across diverse sets of proteins. These difficulties include the wide, potentially confounding range of experimental conditions and methods employed to date and the difficulty of obtaining correct and complete sequence and structural details for the characterized constructs. The lack of a single approach to data analysis and error estimation, or even of a common set of units and reporting standards, further hinders comparative studies of folding. In an effort to overcome these problems, we define here a “consensus” set of experimental conditions (25°C at pH 7.0, 50 mM buffer), data analysis methods, and data reporting standards that we hope will provide a benchmark for experimental studies. We take the first step in this initiative by describing the folding kinetics of 30 apparently two‐state proteins or protein domains under the consensus conditions. The goal of our efforts is to set uniform standards for the experimental community and to initiate an accumulating, self‐consistent data set that will aid ongoing efforts to understand the folding process.

The folding and design of repeat proteins: reaching a consensus

Although they are widely distributed across kingdoms and are involved in a myriad of essential processes, until recently, repeat proteins have received little attention in comparison to globular proteins. As the name indicates, repeat proteins contain strings of tandem repeats of a basic structural element. In this respect, their construction is quite different from that of globular proteins, in which sequentially distant elements coalesce to form the protein. The different families of repeat proteins use their diverse scaffolds to present highly specific binding surfaces through which protein-protein interactions are mediated. Recent studies seek to understand the stability, folding and design of this important class of proteins.

Does trifluoroethanol affect folding pathways and can it be used as a probe of structure in transition states

Nonaqueous co-solvents, particularly 2,2,2-trifluoroethanol (TFE), have been used as tools to study protein folding. By analyzing FKBP12, an α/β-protein that folds with two-state kinetics, we have been able to address three key questions concerning the use of TFE. First, does TFE perturb the folding pathway? Second, can the observed changes in the rate of folding and unfolding in TFE be attributed to a change in free energy of a single state? Finally, can TFE be used to infer information on secondary structure formation in the transition state? Protein engineering experiments on FKBP12, coupled with folding and unfolding experiments in 0% and 9.6% TFE, conclusively show that TFE does not perturb the folding pathway of this protein. Our results also suggest that the changes in folding and unfolding rates observed in 9.6% TFE are due to a global effect of TFE on the protein, rather than the stabilization of any elements of secondary structure in the transition state. Thus, studies with TFE and other co-solvents can be accurately interpreted only when combined with other techniques.

Exploring the folding energy landscape of a series of designed consensus tetratricopeptide repeat proteins

Repeat proteins contain short, tandem arrays of simple structural motifs (20−40 aa). 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. One ubiquitous repeat is the tetratricopeptide motif (TPR), a 34-aa helix-turn-helix motif. In this article we describe the folding kinetics of a series of 7 designed TPR proteins that are assembled from arraying identical designed consensus repeats (CTPRan). These range from the smallest 2-repeat protein to a large 10-repeat protein (≈350 aa). In particular, we describe how the energy landscape changes with the addition of repeat units. The data reveal that although the CTPRa proteins have low local frustration, their highly symmetric, modular native structure is reflected in their multistate kinetics of unfolding and folding. Moreover, although the initial folding of all CTPRan proteins involves a nucleus with similar solvent accessibility, their subsequent folding to the native structure depends directly on repeat number. This corresponds to an increasingly complex landscape that culminates in CTPRa10 populating a misfolded, off-pathway intermediate. These results extend our current understanding of the malleable folding pathways of repeat proteins and highlight the consequences of adding identical repeats to the energy landscape.

Fibrous Nanostructures from the Self‐Assembly of Designed Repeat Protein Modules

Single-protein-chain superhelical filaments are obtained from monomeric repeat proteins by controlling the chemistry and solvent exposure at their terminal interfaces. The assembly was achieved in aqueous solution, at neutral pH value, and at room temperature. The building block was a recombinantly engineered designed tetratricopeptide repeat protein. Directed head-to-tail self-assembly was driven by genetically encoded orthogonal native chemical ligation.

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.

A high-throughput fluorescence chemical denaturation assay as a general screen for protein-ligand binding

Chemical denaturation of ligand-protein complexes can provide the basis of a label-free binding assay. Here, we show how the technique can be used as a sensitive/affordable screen of potential ligands from a pool of lead drug variants. To demonstrate, we characterized the binding of polyketide ligands based on the mTOR inhibitor rapamycin to the cellular immunophilin FKBP12. This used the intrinsic fluorescence of the protein to monitor the chemical denaturation of each FKBP12-ligand complex. The assay was then successfully modified to a 96-well plate-based screen. Both formats were able to differentiate binding affinities across a wide dynamic range.

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale.

Protein stability and ligand‐binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5–20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000‐fold to just 108 molecules. The stability of wild‐type FKBP‐12 and a destabilizing binding‐pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP‐12 and rapamycin originates from residue Phe99 in the binding site.Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10(8) molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

LcrH, a Class II Chaperone from the Type Three Secretion System, Has a Highly Flexible Native Structure

Background: Bacterial pathogens using the type III secretion system (T3SS) require class II chaperones (LcrH) for infection. Results: The modular TPR fold of LcrH produces a weakly folded structure held together by its weak dimeric interface. Conclusion: Under physiological conditions, LcrH populates partially folded structures, thus suggesting a mechanism for cargo binding. Significance: This work is important for understanding one essential part of the T3SS. The type three secretion system is a large and complex protein nano-machine that many Gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called “translocators.” Importantly, effective secretion and thus pore formation of the translocators depend on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats. However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical ultracentrifugation and equilibrium denaturations showed that LcrH is a weak and thermodynamically unstable dimer (KD ≈15 μm, ΔGH2O = 7.4 kcal mol−1). The modular tetratricopeptide repeat structure of the dimer allows it to readily unfold in a noncooperative manner to a one-third unfolded dimeric intermediate (ΔGH2O = 1.7 kcal mol−1), before cooperatively unfolding to a monomeric denatured state (ΔGH2O = 5.7 kcal mol−1). Thus, under physiological conditions, the chaperone is able to populate C-terminally unraveled partially folded states, while being held together by its dimeric interface. Such ability suggests a “fly-casting” mechanism as a route to binding their far larger translocator cargo.