Our Current Focus
HOFMEISTER EFFECTS OF GROUP II CATIONS AS SEEN IN THE UNFOLDING OF RIBONUCLEASE A
This work studies the effects of alkaline-earth cation addition on the unfolding free energy of a model protein, pancreatic Ribonuclease A (RNase A) by differential scanning calorimetry analysis. RNase A was chosen because: a) it does not specifically bind Mg2+, Ca2+ and Sr2+ cations and b) maintains its structural integrity throughout a large pH range. We have measured and compared the effects of NaCl, MgCl2, CaCl2 and SrCl2 addition on the melting point of RNase A.
ION SPECIFIC EFFECTS ON THE FOLDING THERMODYNAMICS OF PARATOX, A GROUP A STREPTOCOCCUS PROPHAGE PROTEIN.
The effect of salts on the conformational changes of intrinsically disordered proteins (IDP) can be understood by electrostatic charge screening as well as nonpolar Hofmeister effects. However, IDPs are usually composed of more uncharged residues that are influenced by the salting-out effect. In this work, we investigate ion specific effects on the folding thermodynamics of Paratox, a group A streptococcus prophage protein, that acts as an IDP in the absence of its macromolecular interaction partners. We have demonstrated the dose dependant effect of co-solutes on protein structure trough Circular dichroism (CD) and HSQC-NMR spectroscopy. The salt-specific effects on the tertiary structure were observed by steady-state florescence spectroscopy using intrinsic fluorescence of a F31W mutant. The ion specific Hofmeister effect (m-value) of Prx, follows the salt dependant micellization free energy increments (MFI) determined from model compounds in previous works. Together, we have demonstrated that salting-out effects of hydrophobic groups has a significant contribution to the folding thermodynamics of Paratox.
INVESTIGATING THE EFFECTS OF VARIOUS CO-SOLVENTS ON PROTEIN FOLDING
Protein folding involves the physical transition of a linear polypeptide chain into its native, biologically active 3-dimensional structure. The native structure is based on the amino acid sequence encoded in its polypeptide chain. However, the folding process also depends on the protein’s microenvironment and other physicochemical conditions. Co-solvents are solvent additives that modulate protein stability by shifting the protein folding equilibrium in aqueous environments. Protecting osmolytes like trimethylamine-N-oxide (TMAO) and xylitol push the equilibrium to favor the native state, while denaturing osmolytes like urea favor the unfolded state. Additionally, some organic solvents (e.g. alcohols) have been shown to induce the folding of disordered peptides into α-helical structures in aqueous solutions. Though the effects of numerous co-solvents have been widely demonstrated throughout the literature, their mechanisms of action remain ambiguous. It would be interesting to determine the nature of these interactions, and whether they follow a mechanism that is direct and/or indirect. In this work, model compounds were used to probe the effects of urea, TMAO, xylitol and various organic solvents on protein folding; using a combination of absorbance and fluorescence spectroscopy.
EFFECT OF VARIOUS SALTS ON THE TETRAMERIZATION OF MELITTIN
Understanding the function of salts on protein’s stability and dynamics has been one of the major interesting fundamental research subjects due to its application in protein purification and crystallography methods in biochemistry and biology studies. Previous studies have shown that salts have major effects on protein stability through modulating electrostatic and hydrophobic interactions. It is also known that salt effects on biomolecular thermodynamics are ion-specific. According to electrostatic theory, only the charge and size of ions cause differences between the properties of ions. However, experimental results show that different salts have various effects on the properties and structure of proteins which depends on the nature of the ions. In this study, we analyze the effects of different salts on the thermodynamics of the tetramerization in our model protein system ‘melittin’. Melittin (honeybee venom) is a cationic hemolytic and amphipathic peptide with 26 amino acid residues. To demonstrate the effect of salts, tetramerization of melittin is a useful model peptide because its structure changes from a random coil monomer to a helical tetramer at high ionic strength.
EFFECT OF SALTS ON ENZYME ACTIVITY: USING RIBONUCLEASE A AS A MODEL SYSTEM
Salts influence the stability and folding of proteins. To date, substantial efforts have been focused on finding mechanisms through which salts affect protein thermodynamics. In contrast, the role of salt ions on enzyme catalysis has received less attention and the underlying mechanisms have remained elusive. Here, we analyze the interactions between different cations and anions with different parts of the enzyme surface, including aliphatic, aromatic, amide, and carboxylate groups in order to figure out how enzyme-substrate reaction is affected by ions. We have measured and compared the effects of several salts on the specificity constant (kcat/KM) of ribonuclease A (RNase A) as a model enzyme.