pH and Charged Mutations Modulate Cold Shock Protein Folding and Stability: A Constant pH Monte Carlo Study.

The folding and stability of proteins is a fundamental problem in several research fields. In the present paper, we have used different computational approaches to study the effects caused by changes in pH and for charged mutations in cold shock proteins from Bacillus subtilis (Bs-CspB). First, we have investigated the contribution of each ionizable residue for these proteins to their thermal stability using the TKSA-MC, a Web server for rational mutation via optimizing the protein charge interactions. Based on these results, we have proposed a new mutation in an already optimized Bs-CspB variant. We have evaluated the effects of this new mutation in the folding energy landscape using structure-based models in Monte Carlo simulation at constant pH, SBM-CpHMC. Our results using this approach have indicated that the charge rearrangements already in the unfolded state are critical to the thermal stability of Bs-CspB. Furthermore, the conjunction of these simplified methods was able not only to predict stabilizing mutations in different pHs but also to provide essential information about their effects in each stage of protein folding.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance

ABSTRACT Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance.

Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis.

The gram-positive bacterium Corynebacterium pseudotuberculosis is the causative agent of different diseases that cause dramatically reduced yields of wool and milk, and results in weight loss, carcass condemnation and also death mainly in sheep, equids, cattle and goats and therefore globally results in considerable economical loss. Cold shock proteins are conserved in many bacteria and eukaryotic cells and they help to restore normal cell functions after cold shock in which some appear to have specific functions at normal growth temperature as well. Cold shock protein A from C. pseudotuberculosis was expressed in Escherichia coli and purified. The thermal unfolding/refolding process characterized by circular dichroism, differential scanning calorimetry and NMR spectroscopy techniques indicated that the refolding process was almost completely reversible.