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.

Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of β-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.

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.

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.

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 β-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.

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.

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.

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.

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.

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.

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.