Long time-scale probing of the protein globular core using hydrogen-exchange and room temperature phosphorescence.

Preliminary room temperature phosphorescence measurements of the highly buried Trp109 in E. coli alkaline phosphatase have been used to report on the kinetics of protein hydrogen-deuterium exchange. Upon dilution in D2O the phosphorescence lifetime increases (at 20 degrees C) in a biphasic manner with an immediate change (< 30 seconds) followed by a slow change occurring on an extremely long timescale (days).

Direct kinetic evidence for triplet state energy transfer from Escherichia coli alkaline phosphatase tryptophan 109 to bound terbium.

The addition of excess Tb3+ to metal-depleted Escherichia coli alkaline phosphatase results in enhanced luminescence from enzyme-bound terbium, which increases with sample deoxygenation and exhibits a tryptophan-like excitation spectrum. Following pulsed excitation at 280 nm, the time-resolved terbium emission shows a negative prefactor associated with a submillisecond rise time, which is independent of the concentration of dissolved oxygen. The absence of a build-up phase and similarity in lifetime in the decay kinetics of directly excited (488 nm) terbium allows for the assignment of the submillisecond component in the 280 nm excited sample to bound terbium.

Nanosecond time-resolved circular polarization of fluorescence: study of NADH bound to horse liver alcohol dehydrogenase.

Circularly polarized luminescence (CPL) spectroscopy provides information on the excited-state chirality of a lumiphore analogous but complementary to information regarding the ground-state chirality derived from circular dichroism. The sensitivity of CPL spectra to molecular conformation makes this technique uniquely suited for the study of biomolecular structure, as extensively demonstrated in earlier studies. Unfortunately, the CPL spectra of many biomolecules often contain significantly overlapping contributions from emitting species either because multiple lumiphores are present (e.

Lack of correspondence between the room-temperature phosphorescence decay-components and Trp residues in a series of Trp-->Cys or Trp-->Phe mutants of human carbonic anhydrase II.

The room temperature phosphorescence of native human carbonic anhydrase (CA), and several mutants of this enzyme has been investigated. In these mutants the seven tryptophan residues in the native protein have sequentially been replaced by cysteine or phenylalanine. All of the mutants as well as native CA show room-temperature tryptophan phosphorescence (RTP) spectra.

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets.

A comparative investigation of snake venom neurotoxins and their triplet-state tryptophan-disulfide interactions using phosphorescence and optically detected magnetic resonance.

We have investigated the luminescence and optically detected magnetic resonance (ODMR) of the highly homologous snake venom neurotoxins alpha-bungarotoxin (BgTX), alpha-cobratoxin (CbTX), and cobrotoxin (CoTX) in frozen aqueous glasses. The phosphorescence intensity and lifetime of the single invariant tryptophan, Trp29, are found to be diminished in BgTX and CbTx relative to CoTX both at 77 K and at 4.2 K.

alpha-Bungarotoxin binding to two acetylcholine receptor alpha-peptides and their methylmercury-modified analogs: intrinsic phosphorescence and optically detected magnetic resonance studies.

Phosphorescence and optically detected magnetic resonance (ODMR) have been used to characterize two synthetic peptides, alpha 181-198 and alpha 185-196, of the major binding determinant of the alpha-acetylcholine receptor (AChR) of Torpedo californica and its interaction with alpha-bungarotoxin (BgTX) using Trp as an intrinsic probe. BgTX conformational changes are suggested upon complexation with the peptides. Methylmercury-modified peptides show conformational heterogeneity which brings some of the modified Cys residues into proximity of peptide Trp(s).