| Cytochrome c 1. Hydrophobic interaction of 8-Anilino-1-Napthalenesulfonic Acid (ANS) with proteins is one of the widely used methods for characterization/detecting partially folded states of proteins. We carried out a systematic investigation on the effects of ANS, a charged hydrophobic fluorescent dye, on conformational properties of acid-unfolded horse cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectrometry (ESI MS). These studies suggested at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino-acid residues present in acid unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten-globule state. Ali, V: Prakash, K: Kulkarni, S: Ahmad, A: Madhusudanan, K. P and Bhakuni, V. (1999) 8-Anilino-1-Napthalenesulfonic Acid Induces Folding of Acid Unfolded Cytochrome c to Molten Globule State as a Result of Electrostatic Interactions. Biochemistry 38,13635-13642 2. Trichloroacetic acid (TCA) and trifluoroacetic acid (TFA) induced unfolding of horse cytochrome c was followed by using a combination of optical techniques and electrospray ionization mass spectroscopy (ESI MS). The results suggested that the trichloroacetate anion has a greater affinity for cytochrome c compared to trifluoroacetate anion, which might be the probable reason for the stabilization of the native like folded intermediate during TCA induced denaturation of cytochrome c as compared to extensive unfolding observed in presence of TFA. Ahmad, A: Madhusudanan, K. P: and Bhakuni V. (2000) Trichloroacetic Acid and Trifluoroacetic Acid Induced Unfolding of Cytochrome c: Stabilization of a Native like Folded Intermediate. Biochim. Biophys. Acta. 1480, 201-210 Glucose oxidase 1. Glucose oxidase from Aspergillus niger is an acidic dimeric enzyme having high localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations, on the structure, function and stability of glucose oxidase using various spectroscopic techniques, limited proteolysis, enzyme assays, size exclusion chromatography, and differential scanning calorimetry (DSC). The monovalent cations were found to influence the enzyme activity but had no effect on the secondary structure of enzyme. Comparative denaturation studies in the presence and absence of monovalent cations demonstrated significant resistance towards urea denaturation in presence of cations. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister Series i.e., K+ > Na+ > Li+. Ahmad, A: Akhtar, M.S: and Bhakuni, V. (2001) Monovalent Cation Induced Conformational Change in Glucose Oxidase Leading to Stabilization of Enzyme. Biochemistry 40, 1945-1955. 2. In a striking difference to the Monovalent cations the effect of divalent cations on the glucose oxidase revealed a different mechanism of action by these cations. The studies of divalent cations were followed by optical spectroscopy, DSC, Enzyme activity and proteolytic digestions. The Tm and DH obtained from DSC favor the Monomeric glucose oxidase While both the near UV CD and fluorescence show opened up structure at 2M divalent salt concentration. The presence of two minima corresponding to 222nm and 209nm in far UV CD show the secondary structure of the monomers to be intact although a little loosened up. The loss of enzyme activity at fairly low concentration of divalent cations and the observations found by other techniques clearly show that the divalent cations cause unfolding and the inhibition of enzyme activity. Akhtar, M.S: Ahmad, A: and Bhakuni, V. (2002) Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: Dual effect of dimer dissociation with loss of cooperative interactionsin enzyme monomer. Biochemistry 42, 7142-7149. 3. The folding/unfolding behavior of glucose oxidase was followed by optical methods, DSC, limited proteolysis, enzyme activity and gel permeation chromatography using urea and GdnHCl as denaturating agents. GdnHCl denaturation was found to be protein concentration dependent and involving more than one transition from folded to unfolded state. Though Cm and m values did not alter but a marked difference in the activity at lower GdnCl concentration was observed particularly between 0-0.2M GdnHCl. Urea induced denaturation was found to be a simple two state process and did not show any dependence on the protein concentration. Akhtar, MS: Ahmad, A: and Bhakuni, V. (2002) Guanidinium chloride and urea induced unfolding of dimeric enzyme glucose oxidase. Biochemistry 41, 3819-3827. Crystallography: Glucose oxidase (GOD) was purified from the commercial supply. 4-8mg/ml was mixed with reservoir buffer in the solution carrying PEG (3350) 20-26% w/v, ammonium acetate buffer pH 4.5 - 50mM, in 1:1 ratio. 4ul of this solution was used for hanging drop vapor diffusion method against 1ml reservoir solution and the samples were incubated at 4 C. After two weeks crystals of GOD were observed. The method was repeated in presence of GdmCl 0.5M and the crystals were found to grow over two weeks. The Crystals were mounted on Rigaku generator diffraction and resolution in absence of nitrogen current was found to be in the range of 2.5 – 3 nm. Two crystals were used in collecting diffraction data in either condition at room temperature. |