Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching.
We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.
Lakowicz
J R
JR
University of Maryland, School of Medicine, Department of Biological Chemistry, Baltimore 21201.
Gryczynski
I
I
Szmacinski
H
H
Cherek
H
H
Joshi
N
N
D020106:Acrylamide; D000178:Acrylamides; D000324:Adrenocorticotropic Hormone / Q000737:chemistry*; D000595:Amino Acid Sequence; D001718:Birefringence; D005454:Fluorescence Polarization; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry; D010100:Oxygen; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D046911:Macromolecular Substances; D009842:Oligopeptides; D010940:Plant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii; D020106:Acrylamide; D014364:Tryptophan; D000324:Adrenocorticotropic Hormone; D006163:Ribonuclease T1; D008836:Micrococcal Nuclease; D010100:Oxygen