MOTIONAL DYNAMICS OF A BURIED TRYPTOPHAN REVEALS THE PRESENCE OF PARTIALLY STRUCTURED FORMS DURING DENATURATION OF BARSTAR

A double mutant of the single-domain protein barstar having a single t ryptophan (W53) was made by mutating the remaining two tryptophans (W3 8 and W44) into phenylalanines. W53 is buried in the core of barstar. Time-resolved fluorescence of the mutant barstar (W38FW44F) showed tha t W53 has a single fluorescence lifetime in the native (N) state and h as three lifetimes in the molten globule-like low-pH (A) form. Quenchi ng of fluorescence by either KI or acrylamide showed that W53 is solve nt inaccessible in the N-state and fairly accessible in the A-form. Th e denaturation of W38FW44F by guanidine hydrochloride (GdnHCl) was mon itored by several probes: near-UV and far-UV circular dichroism (CD), fluorescence intensity, and steady-state and time-resolved fluorescenc e anisotropy. While the unfolding transitions observed through CD and fluorescence intensity coincided with each other (midpoint approximate to 1.8 M GdnHCl), the transition observed through the steady-state fl uorescence anisotropy was markedly different from others. Initially, t he anisotropy increased with the increase in the concentration of GdnH Cl and decreased subsequently. The midpoint of this titration was 2.2 M GdnHCl. Picosecond time-resolved fluorescence anisotropy showed that W38FW44F has a single rotational correlation time of 4.1 ns in the na tive (N) state and 1.5 ns in the unfolded (U) state (6 M GdnHCl). Thes e could be explained as being due to the absence of motional freedom o f W53 in the N-state and the presence of rotational freedom in the U-s tate. In the intermediate concentration region (1.8-3.0 M GdnHCl), the anisotropy decays showed at least two correlation times, similar to 1 and 6-12 ns, These two correlation times are ascribed to partially st ructured forms leading to hindered rotation of W53. Thus, the usefulne ss of time-resolved fluorescence anisotropy in detecting partially fol ded structures is demonstrated.