Biomedical subjects

B D Schlyer

Publications and source records attributed to B D Schlyer.

7 recordsLinked to original sources

PubMed1996

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). The immediate D2O-induced lifetime increase is similar to that observed upon dilution into glycerol, a known protein hydrating agent. On the other hand, the slow D2O-induced first order growth in Trp109 lifetime is due to exchange at highly protected protein groups. As the phosphorescence lifetime of Trp109 is dependent on local rigidity, this increase in lifetime reflects changes in alkaline phosphatase structure. This first use of room temperature phosphorescence to monitor proton exchange shows promise as a sensitive and selective probe of protein core dynamics.

Alkaline Phosphatase↗

PubMed1995

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. The results of the steady state and time-resolved experiments suggest that the time evolution of alkaline phosphatase-bound terbium emission is determined by energy transfer (kET approximately 360 and 120 s-1) from the triplet state of tryptophan to terbium followed by terbium decay. This model is based on the observations that 1) the tryptophan phosphorescence lifetime (previously assigned to Trp109) corresponds to the longer component of the terbium emission and 2) the long-lived emission is enhanced, as is the Trp109 phosphorescence, by deoxygenation. An energy transfer mechanism involving the Trp109 triplet state is shown to be inconsistent with a dipole-dipole process and is best understood as a through-space electron exchange over a donor-acceptor distance of 9-10 A.

Alkaline Phosphatase↗

PubMed1995

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.g., tryptophan residues in a protein) or because multiple conformations of the biomolecule simultaneously exist, each with a unique CPL spectrum. Increased resolution between individual contributions to the CPL may be achieved by time-resolving this signal, thus taking advantage of the fact that, as a rule, each of the emitting species also has a characteristic decay time associated with its electronically excited state. In addition, the time resolution provides information regarding dynamics associated with the different chiral states of the system. The present study describes an instrument for the determination of time-resolved CPL (TR-CPL) with subnanosecond resolution and its application to several chiral systems. The technique was first demonstrated on a model system with a strong time-dependent CPL signal. Subsequently, the circularly polarized component in the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) bound to liver alcohol dehydrogenase was time-resolved. The CPL of NADH in the binary enzyme-coenzyme complex is time-dependent, reflecting structural differences around the reduced nicotinamide possibly due to a dynamic restructuring. In contrast, the CPL of the coenzyme in the ternary complex formed with enzyme and the substrate analog isobutyramide is essentially time-independent, likely reflecting a more rigid binding domain. Since the linear polarization of the fluorescence of the two complexes did not show any local flexibility of the NADH chromophore, the excited-state conformational rearrangement of the binary complex indicates a subtle change in its interactions with group(s) in direct contact with it.

Alcohol Dehydrogenase↗

PubMed1994

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. Surprisingly, only small differences in RTP life-times are noticeable among these mutants, indicating that there is more than one tryptophan residue with similar phosphorescence decay kinetics in the protein. The present results illustrate the danger in attributing the room temperature phosphorescence of a multi-tryptophan protein to a particular residue based solely on an analysis of the protein structure.

Carbonic Anhydrases↗

PubMed1994

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. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.

Alcohol Dehydrogenase↗

PubMed1992

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. Selective reduction of the Cys30-Cys34 disulfide proximal to Trp29 in BgTX and CbTX, that is absent in CoTX, results in the enhancement of the phosphorescence to fluorescence intensity ratio of Trp29 and identifies this disulfide as the source of the triplet-state quenching. Variations of the phosphorescence parameters are observed for differently frozen BgTX and CbTX samples. We argue that this observation is consistent with conformational flexibility in the region of Trp29. For BgTX and CbTX, changing the wavelength of excitation from 285 to 300 nm results in a small bathochromic phosphorescence shift of 0.4 nm, an average decrease in the lifetime, and a change in the polarity of the normally positive D-E ODMR signal. From the small excitation-dependent emission shift, we infer that Trp29 is in a relatively hydrophobic environment. The excitation-dependent changes in lifetime and ODMR signal parameters arise from subtle heterogeneity in the disposition of Trp29 with respect to Cys30-Cys34. We discuss the mechanism of disulfide-induced quenching of the Trp29 triplet state in BgTX and CbTX and argue that it most probably is due to electron transfer.

Amino Acid Sequence↗

PubMed1992

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). These modified peptides, when bound to BgTX, undergo structural changes which remove the tagged Cys from its close contact with the Trp residue(s) of the peptide.

Amino Acid Sequence↗