Protein & Peptide Letters     Monika A. Coronado a,b , Icaro P. Caruso b , Vinícius M. de Oliveira b , Vinícius G. Contessoto b , Vitor B.P. Leite b , Liege A. Kawai a,b , Raghuvir K. Arni a,b and Raphael J. Eberle a,b, * a Multiuser Center for Biomolecular Innovation, Universidade Estadual Paulista (UNESP), São Jose do Rio Preto-SP, 15054-000, Brazil; b Department of Physics, Universidade Estadual Paulista (UNESP), São Jose do Rio Preto-SP, 15054-000, Brazil A R T I C L E H I S T O R Y Received: November 30, 2016 Revised: January 25, 2017 Accepted: January 25, 2017 DOI: 10.2174/0929866524666170207 153808 Abstract: The conformational stability of the Cold shock protein A (CspA) from C. pseudotubercu- losis (Cp), a nucleic acid binding protein in function of pH and salt concentration was examined by using differential scanning calorimetry and CD spectroscopy in combination with computational analysis to identify the specify amino acids undergoing change. Our approach identified a sodium- binding site in CpCspA and at pH 8.0 a significant reduction in the β-sheet content was observed which resulted in a decrease of the protein thermal stability. The computational analyses identified His30 and His65 as the amino acids with the largest charge shifts at different pHs. His30/His65 are part of the extensive hydrogen bonding network and along with the ion-binding site are essential for the conformational stability of CspA. Keywords: Cold shock protein, C. pseudotuberculosis, ion binding, histidine, pH, secondary structure. 1. INTRODUCTION Bacteria respond to changes in environmental parameters such as nutrient levels, oxygen availability, osmotic stress and temperature by triggering the activation of a specific set of genes that up-regulate the production of cold shock pro- teins (Csps) and consequently, by restricting the production of non-cold-shock proteins [1-3]. The initial phase of the cold shock response, the acclima- tion phase, is characterized by high levels of expression of Csp which stimulates the synthesis of other cold-stress re- lated proteins [4, 5]. At the end of the acclimation phase the synthesis of the cold shock proteins is diminished and the levels of non-cold shock protein synthesis are reestablished [6]. The primary function of Csps involves the enhancement of DNA transcription to support the expression of other cold shock induced genes [5, 7, 8]. This is achieved by the high affinity and specificity of Csps in binding to short single- stranded DNAs [9-12]. The observed non-specific binding of Csps to ssRNA suggests that they could also function as RNA chaperones [13] by preventing the formation of cold-induced mRNA secondary structures [14]. Both mecha- nisms help to preserve cell viability during the cold shock process and also in restoring normal cell functions. *Address correspondence to this author at the Multiuser Center for Bio- molecular Innovation, Universidade Estadual Paulista (UNESP), Rua Cristóvão Colombo 2265, São Jose do Rio Preto-SP, 15054-000, Brazil; Tel/Fax: +55-17-32212707, +55-17-32212247; E-mail: eberleraphael@gmail.co Csps are encountered in a wide range of bacterial species, such as psychrophilics, mesophilics, thermophilics and hy- perthermophilics and share highly conserved sequences and structural motifs. The three-dimensional structures of a num- ber of Csps have been determined, including the E. coli CspA, B. subtilis CspB, and B. caldolyticus CspB, and they possess a typical β-barrel fold, consisting of a three stranded N-terminal sheet (β1-β2-β3) and a two stranded C-terminal sheet (β4-β5). Csps contain two nucleic acid binding motifs (RNP1 and RNP2) consisting mainly of aromatic and basic amino acid residues that participate in the binding to single- stranded nucleic acids [15-18]. Despite the high degree of conservation of Csps se- quences and principal structural motifs, the thermal stability of these proteins varies significantly; the E. coli CspA un- folds at 60 °C [16, 19], whereas the B. subtilis CspB unfolds at 52 °C [10]. The melting temperature (T m ) of the thermo- philic B. caldolyticus Csp is 77 °C [17], twenty-five degrees higher than the T m of B. subtilis CspB, and the reversible unfolding process is a monomolecular (N) ↔ unfolded (U) two state reaction [20]. Theoretical and experimental inves- tigations on the thermal stability of Csps have examined the role of point mutations [21], variations of salt concentration [17, 22] and pH [23, 24]. Mycobacterium, Nocardia, and Rhodococcus), a cluster of gram-positive bacteria species [25]. C. pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA), a disease encountered in sheep, goats, and equids (ulcerative lymphangitis) and cattle (cutaneous excoriated granulomas) which results in drastically reduced yields of wool and milk, 1875-5305/17 $58.00+.00 © 2017 Bentham Science Publishers Send Orders for Reprints to reprints@benthamscience.ae 358 Protein & Peptide Letters, 2017, 24, 358-367 RESEARCH ARTICLE Cold Shock Protein A from Corynebacterium pseudotuberculosis: Role of Electrostatic Forces in the Stability of the Secondary Structure