Tryptophan phosphorescence as a monitor of protein conformation in molecular films.
This report enquires on the potentiality of Trp phosphorescence for probing the conformational state of proteins deposited on solid dry films. Thin, amorphous protein films were fabricated with Apoazurin, alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase the protein being incorporated into a DEAE-dextran matrix and deposited on quartz slides. The results, obtained with appositely constructed instrumentation, demonstrate that thanks to the low background radiation associated with long-lived, delayed emission phosphorescence can be readily detected down to single protein layer matrices and that both spectrum and lifetime are important indicators of the integrity of the protein globular fold. In fact, denaturation of the proteins by guanidinium hydrochloride or heat treatment points out that disruption of the native fold leads to a red shift and broadening of the spectrum with loss of vibronic structure, accompanied to considerably shorter-lived and more heterogeneous decay kinetics. It is also shown that the sensitivity of the phosphorescence lifetime towards the detection of altered, looser conformations of the polypeptide are remarkably enhanced on partial hydration of the sample.
Strambini
E G
EG
CNR, Istituto di Biofisica, Ghezzana, Pisa, Italy. strambin@ib.pi.cnr.it
Strambini
G B
GB
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000818:Animals; D015374:Biosensing Techniques; D004800:Enzymes, Immobilized / Q000737:chemistry; D005969:Glutamate Dehydrogenase / Q000737:chemistry; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D019791:Guanidine; D066298:In Vitro Techniques; D008163:Luminescent Measurements; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D004800:Enzymes, Immobilized; D014364:Tryptophan; D000426:Alcohol Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005969:Glutamate Dehydrogenase; D019791:Guanidine