Acrylamide quenching of Trp phosphorescence in liver alcohol dehydrogenase: evidence of gated quencher penetration.
Notwithstanding the relevance of their biological function, slow motions in proteins, beyond the microsecond range, are still poorly understood and often elusive. We propose that acrylamide quenching of Trp phosphorescence of deeply buried residues, when extended over the entire accessible range of lifetime measurements (tau > 10 micros), may help to unveil low-frequency protein motions that allow penetration of solute into the protein interior. The work examines in some detail acrylamide quenching of Trp phosphorescence in a model protein (liver alcohol dehydrogenase) over an extended submillimolar to molar acrylamide concentration range. The results, which encompass a >10(4)-fold variation in the quenching rate, provide the first evidence of a downward-curving lifetime Stern-Volmer plot, indicative of a nonlinear dependence of the quenching rate on the quencher concentration. From an analysis of saturation effects in terms of a protein-gated acrylamide diffusion mechanism, we infer two main routes for acrylamide to penetrate the globular fold and come into the proximity of internal W314: a low-frequency gate [36 s(-1) (at 25 degrees C)] tentatively assigned to partial opening of the dimer interface and a higher-frequency one (11800 s(-1)) tentatively assigned to a channel blocked by the side chains of V276 and L307. These motions are sharply inhibited in the rigid protein complexes formed with the coenzyme NAD(+) and the coenzyme analogue adenine diphosphate ribose, as well as by the frictional drag of the solvent in viscous glycerol solutions, evidence that rules out an alternative quenching mechanism involving acrylamide binding to the protein.
Strambini
Giovanni B
GB
Consiglio Nazionale delle Ricerche, Istituto di Biofisica, Pisa, Italy. strambini@pi.ibf.cnr.it
Gonnelli
Margherita
M
D020106:Acrylamide / Q000737:chemistry / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D018360:Crystallography, X-Ray; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D006736:Horses; D008099:Liver / Q000201:enzymology*; D008163:Luminescent Measurements; D008956:Models, Chemical; D012996:Solutions; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D012996:Solutions; D020106:Acrylamide; D014364:Tryptophan; D000426:Alcohol Dehydrogenase