Thermodynamics of Gndhcl Induced Unfolding of A Helical Membrane Protein in Mixed Micelles

ERNESTO A. ROMAN; JOSé M ARGüELLO; F. LUIS GONZÁLEZ FLECHA

San Francisco, USA

Congreso; 54th Biophysical Society Annual Meeting; 2010

Biophysical Society

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties to find experimental conditions where reversible denaturation could be possible.In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804 residues á-helical membrane protein involved in transporting Cu+ throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far UV ellipticity, and the loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features characteristic of the native enzyme. CopA unfolding showed typical characteristics of a two state process with ÄGw° = 12.9 kJ·mol-1, m= 4.1 kJ·mol-1·M-1, Cm = 3 M and ÄCpw° = 0.93 kJ·mol-1·K-1. The obtained results suggest the existence of non-detectable unfolding intermediates, and point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structure was disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state with a significant spectral red shift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-aniline-8-naphtalenesulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures.