Differential Effects of Hydrophobic Core Packing Residues for Thermodynamic and Mechanical Stability of a Hyperthermophilic Protein.

Proteins from organisms that have adapted to environmental extremes provide attractive systems to explore and determine the origins of protein stability. Improved hydrophobic core packing and decreased loop-length flexibility can increase the thermodynamic stability of proteins from hyperthermophilic organisms. However, their impact on protein mechanical stability is not known. Here, we use protein engineering, biophysical characterization, single-molecule force spectroscopy (SMFS), and molecular dynamics (MD) simulations to measure the effect of altering hydrophobic core packing on the stability of the cold shock protein TmCSP from the hyperthermophilic bacterium Thermotoga maritima. We make two variants of TmCSP in which a mutation is made to reduce the size of aliphatic groups from buried hydrophobic side chains. In the first, a mutation is introduced in a long loop (TmCSP L40A); in the other, the mutation is introduced on the C-terminal ß-strand (TmCSP V62A). We use MD simulations to confirm that the mutant TmCSP L40A shows the most significant increase in loop flexibility, and mutant TmCSP V62A shows greater disruption to the core packing. We measure the thermodynamic stability (ΔGD-N) of the mutated proteins and show that there is a more significant reduction for TmCSP L40A (ΔΔG = 63%) than TmCSP V62A (ΔΔG = 47%), as might be expected on the basis of the relative reduction in the size of the side chain. By contrast, SMFS measures the mechanical stability (ΔG*) and shows a greater reduction for TmCSP V62A (ΔΔG* = 8.4%) than TmCSP L40A (ΔΔG* = 2.5%). While the impact on the mechanical stability is subtle, the results demonstrate the power of tuning noncovalent interactions to modulate both the thermodynamic and mechanical stability of a protein. Such understanding and control provide the opportunity to design proteins with optimized thermodynamic and mechanical properties.

Tuning protein mechanics through an ionic cluster graft from an extremophilic protein.

Proteins from extremophilic organisms provide excellent model systems to determine the role of non-covalent interactions in defining protein stability and dynamics as well as being attractive targets for the development of robust biomaterials. Hyperthermophilic proteins have a prevalence of salt bridges, relative to their mesophilic homologues, which are thought to be important for enhanced thermal stability. However, the impact of salt bridges on the mechanical properties of proteins is far from understood. Here, a combination of protein engineering, biophysical characterisation, single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations directly investigates the role of salt bridges in the mechanical stability of two cold shock proteins; BsCSP from the mesophilic organism Bacillus subtilis and TmCSP from the hyperthermophilic organism Thermotoga maritima. Single molecule force spectroscopy shows that at ambient temperatures TmCSP is mechanically stronger yet, counter-intuitively, its native state can withstand greater deformation before unfolding (i.e. it is mechanically soft) compared with BsCSP. MD simulations were used to identify the location and quantify the population of salt bridges, and reveal that TmCSP contains a larger number of highly occupied salt bridges than BsCSP. To test the hypothesis that salt-bridges endow these mechanical properties on the hyperthermophilic CSP, a charged triple mutant (CTM) variant of BsCSP was generated by grafting an ionic cluster from TmCSP into the BsCSP scaffold. As expected CTM is thermodynamically more stable and mechanically softer than BsCSP. We show that a grafted ionic cluster can increase the mechanical softness of a protein and speculate that it could provide a mechanical recovery mechanism and that it may be a design feature applicable to other proteins.

Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of ß-Barrel Assembly Machinery Polypeptide Transport Associated Domains.

Single-molecule force spectroscopy by atomic force microscopy exploits the use of multimeric protein constructs, namely, polyproteins, to decrease the impact of nonspecific interactions, to improve data accumulation, and to allow the accommodation of benchmarking reference domains within the construct. However, methods to generate such constructs are either time- and labor-intensive or lack control over the length or the domain sequence of the obtained construct. Here, we describe an approach that addresses both of these shortcomings that uses Gibson assembly (GA) to generate a defined recombinant polyprotein rapidly using linker sequences. To demonstrate the feasibility of this approach, we used GA to make a polyprotein composed of alternating domains of I27 and TmCsp, (I27-TmCsp)3-I27)(GA), and showed the mechanical fingerprint, mechanical strength, and pulling speed dependence are the same as an analogous polyprotein constructed using the classical approach. After this benchmarking, we exploited this approach to facilitiate the mechanical characterization of POTRA domain 2 of BamA from E. coli (EcPOTRA2) by assembling the polyprotein (I27-EcPOTRA2)3-I27(GA). We show that, as predicted from the α + ß topology, EcPOTRA2 domains are mechanically robust over a wide range of pulling speeds. Furthermore, we identify a clear correlation between mechanical robustness and brittleness for a range of other α + ß proteins that contain the structural feature of proximal terminal ß-strands in parallel geometry. We thus demonstrate that the GA approach is a powerful tool, as it circumvents the usual time- and labor-intensive polyprotein production process and allows for rapid production of new constructs for single-molecule studies. As shown for EcPOTRA2, this approach allows the exploration of the mechanical properties of a greater number of proteins and their variants. This improves our understanding of the relationship between structure and mechanical strength, increasing our ability to design proteins with tailored mechanical properties.

Life in extreme environments: single molecule force spectroscopy as a tool to explore proteins from extremophilic organisms.

Extremophiles are organisms which survive and thrive in extreme environments. The proteins from extremophilic single-celled organisms have received considerable attention as they are structurally stable and functionally active under extreme physical and chemical conditions. In this short article, we provide an introduction to extremophiles, the structural adaptations of proteins from extremophilic organisms and the exploitation of these proteins in industrial applications. We provide a review of recent developments which have utilized single molecule force spectroscopy to mechanically manipulate proteins from extremophilic organisms and the information which has been gained about their stability, flexibility and underlying energy landscapes.

Towards design principles for determining the mechanical stability of proteins.

The successful integration of proteins into bionanomaterials with specific and desired functions requires an accurate understanding of their material properties. Two such important properties are their mechanical stability and malleability. While single molecule manipulation techniques now routinely provide access to these, there is a need to move towards predictive tools that can rationally identify proteins with desired material properties. We provide a comprehensive review of the available experimental data on the single molecule characterisation of proteins using the atomic force microscope. We uncover a number of empirical relationships between the measured mechanical stability of a protein and its malleability, which provide a set of simple tools that might be employed to estimate properties of previously uncharacterised proteins.

Single-molecule force spectroscopy identifies a small cold shock protein as being mechanically robust.

Single-molecule force spectroscopy has emerged as a powerful approach to examine the stability and dynamics of single proteins. We have completed force extension experiments on the small cold shock protein B from Thermotoga maritima, using a specially constructed chimeric polyprotein. The protein's simple topology, which is distinct from the mechanically well-characterized ß-grasp and immunoglobulin (Ig)-like folds, in addition to the wide range of structural homologues resulting from its ancient origin, provides an attractive model protein for single-molecule force spectroscopy studies. We have determined that the protein has mechanical stability, unfolding at greater than 70 pN at a pulling velocity of 100 nm s(-1). We reveal features of the unfolding energy landscape by measuring the dependence of the mechanical stability on pulling velocity, in combination with Monte Carlo simulations. We show that the cold shock protein has mechanically robust, yet malleable, features that may be important in providing the protein with stability and flexibility to function over a range of environmental conditions. These results provide insights into the relationship between the secondary structure and topology of a protein and its mechanical strength. This lays the foundation for the investigation of the effects of changes in environmental conditions on the mechanical and dynamic properties of cold shock proteins.

Single molecule force spectroscopy using polyproteins.

In recent years single molecule force spectroscopy has emerged as a powerful new tool to explore the mechanical stability and folding pathways of individual proteins. This technique is used to apply a stretching force between two points of a protein, unfolding the protein to an extended state. By measuring the unfolding and folding trajectories of individual proteins, insight can be gained into the physical mechanisms of protein folding. In this tutorial review we introduce the reader to single molecule force spectroscopy using the atomic force microscope (AFM), and explain the two main modes of operation of the AFM for force spectroscopy: force-extension and force-clamp. We introduce the approach of using polyproteins to obtain a clear mechanical fingerprint for monitoring the response of proteins to an applied mechanical force. In addition, we provide an informative and representative review of recent research on proteins using single molecule force spectroscopy. We focus on areas which have made a significant contribution to the single molecule protein folding field and highlight emerging areas of research which have wider implications for the general scientific community.

Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant.

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.

Structure-function studies of an engineered scaffold protein derived from stefin A. I: Development of the SQM variant.

Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein-protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in 'loop 2' allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.

Paralogs of genes encoding metal resistance proteins in Cupriavidus metallidurans strain CH34.

Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34is a well-studied example of a metal-resistant proteobacterium. Genome sequence analysis revealed the presence of a variety of paralogs of proteins that were previously shown to be involved in heavy metal resistance. Which advantage has C. metallidurans in maintaining all these paralogs during evolution? Paralogs investigated belong to the families RND (resistance nodulation cell division) or CHR (chromate resistance). The respective genes were localized by PCR either on one of the two native megaplasmids pMOL28 and pMOL30 of strain CH34, or on its chromosomal DNA. Gene expression was studied by real-time reverse transcriptase PCR and by reporter gene constructs. Genes found to be inducible were disrupted and their contribution to metal resistance measured. When two or three highly related genes were present, usually one was inducible by heavy metals while the other one or two were silent or constitutively expressed. This suggests that C. metallidurans CH34 carries a variety of no longer or not yet used genes that might serve as surplus material for further developments, an advantage that may compensate for the costs of maintaining these genes during evolution.

Identification of a regulatory pathway that controls the heavy-metal resistance system Czc via promoter czcNp in Ralstonia metallidurans.

The CzcCBA cation-proton-antiporter is the most complicated and efficient heavy-metal resistance system known today and is essential for survival of Ralstonia metallidurans at high cobalt, zinc, or cadmium concentrations. Expression of Czc is tightly controlled by the complex interaction of several regulators. Double- and multiple-deletion studies demonstrated that four regulators encoded downstream of the czcCBA operon, CzcD, CzcS, CzcR and the newly identified CzcE, are involved in, but not essential for metal-dependent induction of czc. These proteins control expression of the czcNICBA region from the promoter czcNp. Northern analysis showed that czcDRS was transcribed as czcDR-specific and czcDRS-specific mRNAs. Transcription of czcE occurred independently of czcDRS transcription and was induced by zinc. CzcE is a periplasmic protein as indicated by phoA fusions. CzcE was purified and identified as a metal-binding protein. These data demonstrate that the transport protein CzcD, the two-component regulatory system CzcR, CzcS, and the periplasmic metal-binding protein CzcE exert metal-dependent control of czcNICBA expression via regulation of czcNp activity.

First step towards a quantitative model describing Czc-mediated heavy metal resistance in Ralstonia metallidurans.

Quantitative models were derived to explain heavy metal resistance in Ralstonia metallidurans. A deltaczcA deletion of the gene for the central component of the Co2+/Zn2+/Cd2+ efflux system CzcCBA combined with the expression level of czcCBA as studied with a phi(czcC-lacZ-czcBA) operon fusion demonstrated that CzcCBA was the only prerequisite for resistance to Co2+/Zn2+/Cd2+ at concentrations of 200 microM and above. The cellular content of the CzcA protein (resistance nodulation cell division protein family RND) determined by Western blot was used to model the CzcCBA expression level as the response to various metal concentrations. These data and experimentally determined uptake velocities were used to derive a flow equilibrium model that describes the cytoplasmic content c(i) of the cells as an interaction between cation uptake and CzcCBA-mediated efflux. Alternatively, binding of heavy metals to inactivated R. metallidurans cells was described with a modified Freundlich's equation. The metal content of growing R. metallidurans cells was determined and compared to the predictions of both models. High amounts of zinc precipitates. exclusively formed by living cells, prevented a model validation for zinc. An additional net efflux activity let to lower amounts of cell-bound Co2+ than predicted. The flow equilibrium model described cadmium resistance sufficiently for R. metallidurans growing in the presence of 0.2-1 mM Cd2+. Description of cadmium resistance in early stationary cells requires the binding model in addition to the flow equilibrium model. Thus, it was possible to simulate physiological events in growing cells by quantitative models that are derived from the biochemical data of the interacting transport proteins.