title: Amplitude spectrum of structural fluctuations in proteins from the internal diffusion of solutes of increasing molecular size: a Trp phosphorescence quenching study.
abstract: The accessibility of O(2), acrylamide, and four acrylamide derivatives of increasing molecular size {N-(hydroxymethyl)acrylamide, N,N'-methylene-bisacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid} to buried Trp residues in four proteins, as determined by dynamic quenching of their phosphorescence emission, was utilized for probing the amplitude range of structural fluctuations in these macromolecules. The quenching rate constant of each solute, k(q), was determined (at 25 and -5 °C) for liver alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, azurin, and alkaline phosphatase. The results show that high-frequency small amplitude motions pervade the protein globular fold, permitting relatively unhindered diffusion of small diatomic molecules all the way to compact cores of the macromolecule. For larger solutes, the access to deep regions drops sharply with molecular size, with acrylamide probably representing a threshold for diffusion of a solute through homogeneous compact domains, on the long second time scale. The results emphasize the variability in the amplitude of protein motions between deep cores and more superficial regions of the globular fold and unveil the existence of unexpectedly large amplitude low-activation barrier fluctuations permitting the penetration of solutes with comparatively large M(w) values.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56124 Pisa, Italy. strambini@pi.ibf.cnr.it
authors.forename: Giovanni B
authors.initials: GB
authors.lastname: Strambini
authors.forename: Margherita
authors.initials: M
authors.lastname: Gonnelli
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000737:chemistry; D001400:Azurin / Q000737:chemistry; D004058:Diffusion; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D007700:Kinetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D011506:Proteins; D012996:Solutions; D001400:Azurin; D020106:Acrylamide; D014364:Tryptophan; D000426:Alcohol Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000469:Alkaline Phosphatase