Unusual denaturation trajectory of bovine gamma globulin studied by fluorescence correlation spectroscopy

Abstract

Non-native and denatured states of proteins have received increasing attention because of their relevance to issues such as protein folding and stability. In this context, the pathway of polypeptide collapse and random coil formation in a denatured protein is a subject of much interest. Most proteins so far studied have shown monotonic expansion of their hydrodynamic radius (RH) in the presence of increasing concentration of chaotropes. We have studied GdnHCl-induced folding transitions and conformational states of a multi-domain protein, bovine gamma globulin, using fluorescence, circular dichroism and fluorescence correlation spectroscopy (FCS). FCS measurements showed that for gamma globulin, contrary to the observed trend, RH decreases with increasing GdnHCl concentration up to 3 M. At higher GdnHCl concentration, RH starts to increase but exhibits complicated behavior in the form of two sharp maxima at 4 M and 7 M. Further experiments suggest that the maximum at 4 M GdnHCl arises due to electrostatic interaction, whereas the one at 7 M GdnHCl corresponds to the usual expanded conformation due to denaturation. Beyond 7 M GdnHCl, RH decreases drastically and is shown to result from fragmentation of the protein caused by rupture of disulphide bonds by the high GdnHCl concentration. Our results demonstrate the capability of FCS in revealing intricate details of the unfolding trajectory that eludes conventional ensemble techniques such as fluorescence and CD.

A robust spectroscopic method for the determination of protein conformational composition – Application to the annealing of silk

“…Other studies have shown that BSA even in the absence of formal primary β structure does give infrared spectra consistent with significant (~30%) β aggregate nature under denaturing conditions of elevated temperatures, increased ionic strength and drying [62,63], in general agreement with our findings. By contrast, the β sheet content found here for γ globulin is lower than reported from X-ray analysis and circular dichroism [64,65]. Which may be due to our drying process being too rapid for thermodynamic relaxation of the structure but may also be a function of the propensity of globular proteins to form intra-molecular β sheet structure in preference to extended inter-molecular β aggregates [66].…”

Measuring Size, Size Distribution, and Polydispersity of Water-in-Oil Microemulsion Droplets using Fluorescence Correlation Spectroscopy: Comparison to Dynamic Light Scattering

“…Unlike light scattering techniques, fluorescence correlation spectroscopy (FCS) measures the molecular diffusion by correlating fluorescence fluctuations originating from a single (or few) diffusing fluorescent molecule (or molecular aggregate) in and out of a tiny observation volume (∼1 fL) created inside the sample solution using a laser, a high numerical aperture objective lens, and a confocal pinhole placed at the image-plane. − The uniqueness of FCS in measuring diffusion, and subsequently the size and size distribution of particles, lies with the fact that it captures the diffusion time of individual particles in extremely dilute conditions (down to single molecule level), and provides accurate size information on particles in solution. − Moreover, if the fluorescence fluctuations originate from chemical reactions of the observing molecules then one can also extract the kinetic information on such reaction from FCS data. , Because of these advantages, FCS is extensively used to study diffusion and molecular interactions in biomolecular systems. − …”

Efficient and easily scalable protein folding strong anion exchange chromatography for renaturation and simultaneous purification of recombinant human asparaginase from E. coli

“…The other buffers such as sulfate and citrate are next to the phosphate and acetate. However, the less recovery yields owing to the aggregation of protein molecules, which is caused by the intermolecular disulfide bond formation and reduced the recovery yield . The acetate has shown less recovery yield when compared with the other buffers and this could be due to the partial effect of molecular interactions (repulsive forces) between the buffer molecules and protein.…”