Loops linking secondary structure elements affect the stability of molten globule intermediate state of apomyoglobin

Abstract

Apomyoglobin is a protein widely used as a model for studying globular protein folding. This work aimed to test the hypothesis on influence of rigidity and length of loops linking protein secondary structure elements on the stability of molten globule intermediate state. For this purpose, we studied folding/unfolding of mutant apomyoglobin forms with substitutions of proline residues to glycine and with loops elongated by three and six glycine residues. For all the protein forms, denaturation/renaturation kinetic curves at different urea concentrations were obtained, folding/unfolding constants were calculated and dependencies of rate constant logarithms on urea concentrations were plotted. All the data gave an opportunity to calculate free energies of different apomyoglobin states. As a result, the mutations in apomyoglobin loops were demonstrated to have a real effect on intermediate state stability compared to unfolded state. Keywords: apomyoglobin, protein folding, molten globule intermediate state, tryptophan fluorescence, chevron plot, energy profile.of mutants and wild-type protein evidence that the introduced amino acid substitutions had literally no effect on the structure of apomyoglobin (spectra not shown).Kinetic measurements were taken using a spectrofluorimeter Chirascan Spectrometer (Applied Photophysic, UK) equipped with a stopped-flow attachment.The excitation wavelength was 280 nm, and emission spectra were recorded using a 320-nm cut-off glass optical filter. The initial urea concentration was 5.5 M for refolding experiments, and 0.0 M for unfolding ones. The initial protein solution was mixed (1:1) with a buffer of various urea concentrations using the stopped-flow attachment. The final protein concentration was 0.03 mg/mL. All the measurements were carried out at 11˚C in 20 mM sodium phosphate buffer, pH = 6.2.Denaturation/renaturation kinetic curves at different urea concentrations (0-5.5M) were obtained for each protein variant. The technique of the measurements was described in detail in our previous works [2,[6][7][8][9].