Fluorescence quenching of buried Trp residues by acrylamide does not require penetration of the protein fold.
The accessibility of acrylamide to buried Trp residues in proteins, as attested by dynamic quenching of their fluorescence emission, is often interpreted in terms of migration of the quencher (Q) through the globular fold. The quencher penetration mechanism, however, has long been debated because, on one hand, solutes the size of acrylamide are not expected to diffuse within the protein matrix on the nanosecond time scale of fluorescence and, on the other hand, alternative reactions pathways where Q remains in the solvent cannot be ruled out. To test the Q penetration hypothesis, we compared the quenching rates of acrylamide analogs of increasing molecular size (acrylonitrile, acrylamide, and bis-acrylamide) on the buried Trp residues of RNaseT1 and parvalbumin. The results show that the largest molecule, bis-acrylamide, is also the most efficient quencher and that in general the quenching rate is not correlated to quencher size, as expected for a penetration mechanism. Whereas these results rule out significant internal Q migration in the times of fluorescence, it is also demonstrated that up to a depth of burial of 3 A, through-space interactions with acrylamide in the solvent satisfactorily account for the small rate constants reported for these proteins. More generally, this analysis emphasizes that reduced dynamic quenching of protein fluorescence by acrylamide rather than reporting on the structural rigidity of the globular fold reflects the distance of closest approach between the internal chromophore and Q in the aqueous phase.
Strambini
Giovanni B
GB
Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56124 Pisa, Italy. strambini@pi.ibf.cnr.it
Gonnelli
Margherita
M
D020106:Acrylamide / Q000737:chemistry*; D000818:Animals; D029941:Fish Proteins / Q000737:chemistry; D005399:Fishes; D005453:Fluorescence; D010320:Parvalbumins / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D006163:Ribonuclease T1 / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D029941:Fish Proteins; D010320:Parvalbumins; D011506:Proteins; D020106:Acrylamide; D014364:Tryptophan; D006163:Ribonuclease T1