Exploring the 'N-terminal arm' & 'Convex surface' Binding Interfaces of the T3SS Chaperone-Translocator Complexes from P. Aeruginosa.

One infection method widely used by many gram-negative bacteria involves a protein nanomachine called the Type Three Secretion System (T3SS). The T3SS enables the transportation of bacterial "toxins" via a proteinaceous channel that directly links the cytosol of the bacteria and host cell. The channel from the bacteria is completed by a translocon pore formed by two proteins named the major and minor translocators. Prior to pore formation, the translocator proteins are bound to a small chaperone within the bacterial cytoplasm. This interaction is crucial to effective secretion. Here we investigated the specificity of the binding interfaces of the translocator-chaperone complexes from Pseudomonas aeruginosa via the selection of peptide and protein libraries based on its chaperone PcrH. Five libraries encompassing PcrH's N-terminal and central α-helices were panned, using ribosome display, against both the major (PopB) and minor (PopD) translocator. Both translocators were shown to significantly enrich a similar pattern of WT and non-WT sequences from the libraries. This highlighted key similarities/differences between the interactions of the major and minor translocators with their chaperone. Moreover, as the enriched non-WT sequences were specific to each translocator, it would suggest that PcrH can be adapted to bind each translocator individually. The ability to evolve such proteins indicates that these molecules may provide promising anti-bacterial candidates.

Exploring the "N-Terminal Anchor" Binding Interface of the T3SS Chaperone-Translocator Complexes from P. aeruginosa.

The type III secretion system is a large multiprotein complex that many Gram-negative bacteria use for infection. A crucial part of the complex is its translocon pore formed by two proteins: the major and minor translocators. The pore completes a proteinaceous channel from the bacterial cytosol through the host cell membrane and allows the direct injection of bacterial toxins. Effective pore formation is predicated by the translocator proteins binding to a small chaperone within the bacterial cytoplasm. Given the vital role of the chaperone-translocator interaction, we investigated the specificity of the "N-terminal anchor" binding interface present in both translocator-chaperone complexes from Pseudomonas aeruginosa. Isothermal calorimetry (ITC), alanine scanning, and the selection of a motif-based peptide library using ribosome display were used to characterize the major (PopB) and minor (PopD) translocator interactions with their chaperone PcrH. We show that 10 mer PopB51-60 and PopD47-56 peptides bind to PcrH with a KD of 148 ± 18 and 91 ± 9 µM, respectively. Moreover, mutation to alanine of each of the consensus residues (xxVxLxxPxx) of the PopB peptide severely affected or completely abrogated binding to PcrH. When the directed peptide library (X-X-hydrophobic-X-L-X-X-P-X-X) was panned against PcrH, there was no obvious convergence at the varied residues. The PopB/PopD wild-type (WT) sequences were also not prevalent. However, a consensus peptide was shown to bind to PcrH with micromolar affinity. Thus, selected sequences were binding with similar affinities to WT PopB/PopD peptides. These results demonstrate that only the conserved "xxLxxP" motif drives binding at this interface.

Decoupling a tandem-repeat protein: Impact of multiple loop insertions on a modular scaffold.

The simple topology and modular architecture of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats makes them straightforward to dissect and redesign. Repeat-protein stability can be manipulated in a predictable way using site-specific mutations. Here we explore a different type of modification - loop insertion - that will enable a simple route to functionalisation of this versatile scaffold. We previously showed that a single loop insertion has a dramatically different effect on stability depending on its location in the repeat array. Here we dissect this effect by a combination of multiple and alternated loop insertions to understand the origins of the context-dependent loss in stability. We find that the scaffold is remarkably robust in that its overall structure is maintained. However, adjacent repeats are now only weakly coupled, and consequently the increase in solvent protection, and thus stability, with increasing repeat number that defines the tandem-repeat protein class is lost. Our results also provide us with a rulebook with which we can apply these principles to the design of artificial repeat proteins with precisely tuned folding landscapes and functional capabilities, thereby paving the way for their exploitation as a versatile and truly modular platform in synthetic biology.

Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins.

Engineering proteins to assemble into user-defined structures is key in their development for biotechnological applications. However, designing generic rather than bespoke solutions is challenging. Here we describe an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. Three types of component are used: two complementary oligomeric "half-cage" protein fusions and an extendable monomeric "linker" fusion. All are composed of modular protein domains chosen to fulfill the required geometries, with two orthogonal pairs of split intein halves to drive assembly when mixed. This combination enables both one-pot construction of two-component cages and stepwise assembly of larger three-component scalable cages. To illustrate the system's versatility, trimeric half-cages and linker constructs comprising consensus-designed repeat proteins were ligated in one-pot and stepwise reactions. Under mild conditions, rapid high-yielding ligations were obtained, from which discrete proteins cages were easily purified and shown to form the desired trigonal bipyramidal structures.

Context-Dependent Energetics of Loop Extensions in a Family of Tandem-Repeat Proteins.

Consensus-designed tetratricopeptide repeat proteins are highly stable, modular proteins that are strikingly amenable to rational engineering. They therefore have tremendous potential as building blocks for biomaterials and biomedicine. Here, we explore the possibility of extending the loops between repeats to enable further diversification, and we investigate how this modification affects stability and folding cooperativity. We find that extending a single loop by up to 25 residues does not disrupt the overall protein structure, but, strikingly, the effect on stability is highly context-dependent: in a two-repeat array, destabilization is relatively small and can be accounted for purely in entropic terms, whereas extending a loop in the middle of a large array is much more costly because of weakening of the interaction between the repeats. Our findings provide important and, to our knowledge, new insights that increase our understanding of the structure, folding, and function of natural repeat proteins and the design of artificial repeat proteins in biotechnology.

PyFolding: Open-Source Graphing, Simulation, and Analysis of the Biophysical Properties of Proteins.

For many years, curve-fitting software has been heavily utilized to fit simple models to various types of biophysical data. Although such software packages are easy to use for simple functions, they are often expensive and present substantial impediments to applying more complex models or for the analysis of large data sets. One field that is reliant on such data analysis is the thermodynamics and kinetics of protein folding. Over the past decade, increasingly sophisticated analytical models have been generated, but without simple tools to enable routine analysis. Consequently, users have needed to generate their own tools or otherwise find willing collaborators. Here we present PyFolding, a free, open-source, and extensible Python framework for graphing, analysis, and simulation of the biophysical properties of proteins. To demonstrate the utility of PyFolding, we have used it to analyze and model experimental protein folding and thermodynamic data. Examples include: 1) multiphase kinetic folding fitted to linked equations, 2) global fitting of multiple data sets, and 3) analysis of repeat protein thermodynamics with Ising model variants. Moreover, we demonstrate how PyFolding is easily extensible to novel functionality beyond applications in protein folding via the addition of new models. Example scripts to perform these and other operations are supplied with the software, and we encourage users to contribute notebooks and models to create a community resource. Finally, we show that PyFolding can be used in conjunction with Jupyter notebooks as an easy way to share methods and analysis for publication and among research teams.

Programmed Protein Self-Assembly Driven by Genetically Encoded Intein-Mediated Native Chemical Ligation.

Harnessing and controlling self-assembly is an important step in developing proteins as novel biomaterials. With this goal, here we report the design of a general genetically programmed system that covalently concatenates multiple distinct protein domains into specific assembled arrays. It is driven by iterative intein-mediated native chemical ligation (NCL) under mild native conditions. The system uses a series of initially inert recombinant protein fusions that sandwich the protein modules to be ligated between one of a number of different affinity tags and an intein protein domain. Orthogonal activation at opposite termini of compatible protein fusions, via protease and intein cleavage, coupled with sequential mixing directs an irreversible and traceless stepwise assembly process. This gives total control over the composition and arrangement of component proteins within the final product, enabled the limits of the system-reaction efficiency and yield-to be investigated, and led to the production of "functional" assemblies.

Dissecting and reprogramming the folding and assembly of tandem-repeat proteins.

Studying protein folding and protein design in globular proteins presents significant challenges because of the two related features, topological complexity and co-operativity. In contrast, tandem-repeat proteins have regular and modular structures composed of linearly arrayed motifs. This means that the biophysics of even giant repeat proteins is highly amenable to dissection and to rational design. Here we discuss what has been learnt about the folding mechanisms of tandem-repeat proteins. The defining features that have emerged are: (i) accessibility of multiple distinct routes between denatured and native states, both at equilibrium and under kinetic conditions; (ii) different routes are favoured for folding compared with unfolding; (iii) unfolding energy barriers are broad, reflecting stepwise unravelling of an array repeat by repeat; (iv) highly co-operative unfolding at equilibrium and the potential for exceptionally high thermodynamic stabilities by introducing consensus residues; (v) under force, helical-repeat structures are very weak with non-co-operative unfolding leading to elasticity and buffering effects. This level of understanding should enable us to create repeat proteins with made-to-measure folding mechanisms, in which one can dial into the sequence the order of repeat folding, number of pathways taken, step size (co-operativity) and fine-structure of the kinetic energy barriers.

Repeat protein engineering: creating functional nanostructures/biomaterials from modular building blocks.

There is enormous interest in molecular self-assembly and the development of biological systems to form smart nanostructures for biotechnology (so-called 'bottom-up fabrications'). Repeat proteins are ideal choices for development of such systems as they: (i) possess a relatively simple relationship between sequence, structure and function; (ii) are modular and non-globular in structure; (iii) act as diverse scaffolds for the mediation of a diverse range of protein-protein interactions; and (iv) have been extensively studied and successfully engineered and designed. In the present review, we summarize recent advances in the use of engineered repeat proteins in the self-assembly of novel materials, nanostructures and biosensors. In particular, we show that repeat proteins are excellent monomeric programmable building blocks that can be triggered to associate into a range of morphologies and can readily be engineered as stimuli-responsive biofunctional materials.

LcrH, a class II chaperone from the type three secretion system, has a highly flexible native structure.

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 (K(D) ≈15 µm, ΔG(H(2)O) = 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 (ΔG(H(2)O) = 1.7 kcal mol(-1)), before cooperatively unfolding to a monomeric denatured state (ΔG(H(2)O) = 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.

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.

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 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.

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 (approximately 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.

A recurring theme in protein engineering: the design, stability and folding of repeat proteins.

Repeat proteins are ubiquitous and are involved in a myriad of essential processes. They are typically non-globular structures that act as diverse scaffolds for the mediation of protein-protein interactions. These excitingly different structures, which arise from tandem arrays of a repeated structural motif, have generated significant interest with respect to protein engineering and design. Recent advances have been made in the design and characterisation of repeat proteins. The highlights include re-engineering of binding specificity, quantitative models of repeat protein stability and kinetic studies of repeat protein folding.

A new folding paradigm for repeat proteins.

The folding/unfolding transitions of a series of designed consensus tetratricopeptide repeat proteins are quantitatively described by the classical one-dimensional Ising model, which thus represents a new folding paradigm for repeat proteins. Moreover, for the first time for any protein, a theoretical model predicts the folding/unfolding transition midpoint and the width of the transition.

Local and long-range stability in tandemly arrayed tetratricopeptide repeats.

The tetratricopeptide repeat (TPR) is a 34-aa alpha-helical motif that occurs in tandem arrays in a variety of different proteins. In natural proteins, the number of TPR motifs ranges from 3 to 16 or more. These arrays function as molecular scaffolds and frequently mediate protein-protein interactions. We have shown that correctly folded TPR domain proteins, exhibiting the typical helix-turn-helix fold, can be designed by arraying tandem repeats of an idealized TPR consensus motif. To date, three designed proteins, CTPR1, CTPR2, and CTPR3 (consensus TPR number of repeats) have been characterized. Their high-resolution crystal structures show that the designed proteins indeed adopt the typical TPR fold, which is specified by the correct positioning of key residues. Here, we present a study of the thermodynamic properties and folding kinetics of this set of designed proteins. Chemical denaturation, monitored by CD and fluorescence, was used to assess the folding and global stability of each protein. NMR-detected amide proton exchange was used to investigate the stability of each construct at a residue-specific level. The results of these studies reveal a stable core, which defines the intrinsic stability of an individual TPR motif. The results also show the relationship between the number of tandem repeats and the overall stability and folding of the protein.

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 degrees 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.

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.