Study of the non-covalent molecular complexes in gas phase by electrospray ionization mass spectrometry (ESI-MS) represents a promising strategy to probe the intrinsic nature of these complexes. ESI-MS investigation of a series of synthetic octapeptides containing six alanine and two lysine residues differing only by their positions showed the formation of non-covalent dimers, which were preserved in the gas phase. Unlike the monomers, the dimers were found to show only singly protonated state. The decrease in the solvent polarity from water to alcohol showed enhanced propensity of formation of the dimer indicating that the electrostatic interaction plays a crucial role to stabilize the dimer. Selective functionalization studies showed that ε-NH(2) of lysine and C-terminal amide (-CONH(2)) facilitate the dimerization through intermolecular hydrogen bonding network.

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Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.

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The unfolding of the membrane protein, cytochrome c oxidase (CcO) induced by ionic surfaetants have been studied by using circular dichroism, optical absorbance and time resolved tryptophan fluorescence spectroscopic methods. Ionic surfactant cetyltrimethyl ammonium bromide (CTAB) was found to cause denaturation of this membrane protein leading to release of both, the heme a residues from CcO indicated by both CD and optical titration. Upon dissociation of the hemes from the protein matrix; the tryptophan fluorescence intensity of CcO increased drastically and the fluorescence lifetimes became much longer compared to the short lifetimes observed in the native protein. The shortest lifetime of 70 ps observed in the native protein due to strong quenching (energy transfer) of the beme groups, increased ~ 10-fold in the CTAB-unfolded protein indicating complete removal of the heme groups from the protein matrix. Remarkable differences were observed between the mode of actions of ionic surfactants and the commonly used denaturant guanidine hydrochloride. Improved data analysis of maximum entropy method showed that the lifetime distribution pattern in the two cases of unfolding were very different. The lifetimes in guanidine hydrochloride unfolded CcO were much shorter and more widely distributed indicating that the hemes are probably not separated away from the protein matrix and that the unfolded state is highly heterogeneous. Our results further showed that the lauryl maltoside inhibits denaturation of CcO by the ionic surfactant and the initial step of the denaturation possibly involves quantitative replacement of the lauryl maltoside by the ionic surfactant at the surface of the enzyme.