Single molecule studies of immobilized molecules

Abe and co-workers [ 1 ] have put this principle to very good use in the study of the oligomeric state of the H/K-ATPase ion channel. A large body of evidence indicates that the functional form of this enzyme in its native membrane is oligomeric and the nature of this oligomer is of importance in understanding the function of the channel. H/K-ATPase is composed of an a and a P chain and is 

A very elegant demonstration of the functional detail that can be obtained from single molecule studies of immobilized molecules was given by Ha et al. [10]. Their system of interest involves a helicase—a protein which binds to double-stranded DNA and unwinds the helical structure. Helicases are protein motors that are driven by the binding and hydrolysis of ATP. Some helicases function as hexameric rings but the fundamental form of other helicases is a subject of debate, which is addressed in this paper. Previously, bulk studies had been able to 

By addition of a suitable amount of a chemical denaturant (in this case guani-dinium hydrochloride GdnFFCl) the protein can be made to folly unfold or can be poised at the mid-point of a transition determined by an ensemble equilibrium denaturation experiment. Rhoades recorded the histograms of FRET efficiency for the protein under native, denaturing and mid-point conditions (this was determined to be 0.55 M GdnFlCl for adenylate kinase under these conditions). These data indicate a FRET efficiency of 0.8 in the folded state and 0.14 in the folly denatured state (2 M GdnHCl). At a GdnHCl concentration of 0.4 M the presence of two sub-populations was detected. At this concentration of denaturant the protein will spend an almost equal amount of time in the folded and unfolded 

This sophisticated discussion of protein pathways over a complex energy landscape does rely on some assumptions, such as there being no interaction at all between the protein and the liposome and that the dyes themselves cannot adopt 

Steven Chu s [21-23] group have generated an impressive body of work around single molecule studies of the folding and catalytic activity of the ribozymes. Ribozymes are protein-independent RNA enzymes. Like their protein-based 

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