Probing the denatured state ensemble with fluorescence

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Title: Probing the denatured state ensemble with fluorescence
Author: Alston, Roy Willis
Abstract: To understand protein stability and the mechanism of protein folding , it is essential that we gain a better understanding of the ensemble of conformations that make up the denatured state of a protein . The primary goal of the research described here was to see what we might learn about the denatured state using fluorescence . To this end , tryptophan was introduced at five sites in Ribonuclease Sa (RNase Sa ) : D1W , Y52W , Y55W , T76W , and Y81W . The fluorescent properties of the denatured states of these five proteins were studied and compared to the fluorescent properties of eight model compounds : N -acetyl -tryptophan -amide (NATA ) , N -acetyl -Ala -Trp -Ala -amide (AWA ) , N -acetyl -Ala -Ala -Trp -Ala -Ala -amide (AAWAA ) , and five pentapeptides based on the sequence around the original tryptophan substitutions in RNase Sa . Regardless of the denaturant , ?max for the proteins and model compounds differed very little , 349 .3 ? 1 .2 nm . However , significant differences were observed in the fluorescence intensity at ?max (IF ) , suggesting that IF is more sensitive to the immediate environment than ?max . The differences in IF are due in part to quenching by neighboring side chains . More importantly , IF was always significantly greater in the protein than in its corresponding pentapeptide , indicating that the protein exerts an effect on the tryptophan , which cannot be mimicked by the pentapeptide models . Acrylamide and iodide quenching experiments were also performed on the model compounds and proteins . Significant differences in the Stern -Volmer quenching constant (KSV ) were also observed between the proteins and between the proteins and their corresponding pentapeptides . Importantly , the KSV for the protein was always less than in its corresponding pentapeptide . These data along with the IF data show that non -local structure in the unfolded state influences tryptophan fluorescence and accessibility . In summary , these and our other studies show that fluorescence can be used to gain a better understanding of the denatured states of proteins .
URI: http : / /hdl .handle .net /1969 .1 /451
Date: 2004-09-30

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Probing the denatured state ensemble with fluorescence. Available electronically from http : / /hdl .handle .net /1969 .1 /451 .

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