Effects of cavity-forming mutations on the internal dynamics of azurin.
The effects of two single-point cavity-forming mutations, F110S and I7S, on the internal dynamics of azurin from Pseudomonas aeruginosa were probed by the phosphorescence emission of Trp-48, deeply buried in the compact hydrophobic core of the macromolecule. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime (tau(0)) whereas more general effects on structural fluctuations were deduced from the phosphorescence acrylamide quenching rate constant (k(q)), which measures the diffusion of the solute through the protein fold. The results show a spectacular, 4-5 orders of magnitude, increase of k(q) emphasizing that large amplitude structural fluctuations permitting acrylamide migration to the protein core have been drastically enhanced in each azurin mutant. The large, 12-15 kcal/mol, decrease in the activation enthalpy associated to k(q) suggests that the rate enhancement is caused, rather than through a generalized increase of protein flexibility, by the elimination of an inner barrier to the diffusion process. According to tau(0) the chromophore environment is more fluid with I7S but strikingly more rigid with F110S, demonstrating that when internal cavities are formed local effects on the mobility at the mutation site are unpredictable. Both tau(0) and k(q) reveal a structure tightening role of bound Cd(2+) that correlates with the increase in stability from apo- to holo-azurin. While these alterations in internal dynamics of azurin do not seem to play a role on electron transfer through the central region, the enhanced migration of acrylamide emphasizes that cavities may be critical for the rapid diffusion of substrates to buried, solvent inaccessible sites of enzymes.
Cioni
Patrizia
P
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Area della Ricerca di Pisa, Pisa, Italy.
de Waal
Ellen
E
Canters
Gerard W
GW
Strambini
Giovanni B
GB
D020106:Acrylamide / Q000737:chemistry; D019943:Amino Acid Substitution; D001400:Azurin / Q000737:chemistry*; D009154:Mutation; D016062:Porosity; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D001400:Azurin; D020106:Acrylamide; D014364:Tryptophan