title: Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles.
abstract: Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue alpha-helical membrane protein that is 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 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 of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =0.93 kJ mol(-1) K(-1). These results 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 structures were 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 redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate 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.
authors.affiliation: Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires-CONICET, Buenos Aires, Argentina.
authors.forename: Ernesto A
authors.initials: EA
authors.lastname: Roman
authors.forename: José M
authors.initials: JM
authors.lastname: Argüello
authors.forename: F Luis
authors.initials: FL
authors.lastname: González Flecha
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis
chemical_list: D003902:Detergents; D026901:Membrane Transport Proteins; D010743:Phospholipids; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases