Structure and dynamics of proteins encapsulated in silica hydrogels by Trp phosphorescence.
This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds.
Gonnelli
Margherita
M
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Area della Ricerca di Pisa, Via Moruzzi 1, San Cataldo, 56100 Pisa, Italy.
Strambini
Giovanni B
GB
D000178:Acrylamides; D020100:Hydrogels / Q000737:chemistry*; D008163:Luminescent Measurements; D044367:Phase Transition; D011506:Proteins / Q000737:chemistry*; D012399:Rotation; D012822:Silicon Dioxide / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D020100:Hydrogels; D011506:Proteins; D012822:Silicon Dioxide; D014364:Tryptophan