Biomedical subjects

D G Steel

Publications and source records attributed to D G Steel.

26 records · Page 2Linked to original sources

PubMed1995

Phosphorescence reveals a continued slow annealing of the protein core following reactivation of Escherichia coli alkaline phosphatase.

When Escherichia coli alkaline phosphatase (AP) is refolded in vitro after extensive denaturation in 6.2 M guanidine hydrochloride, the enzymatic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the protein, monitored by the recovery of the tryptophan phosphorescence lifetime, returns to its characteristic native-like value over several days. Moreover, the protein lability, measured by the rate of inactivation in 4.5 M guanidine hydrochloride, also changes on a time scale much longer than the recovery of activity. These results clearly demonstrate that while the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active conformation, the phosphorescence data indicate otherwise. In the context of the rugged energy landscape model [Frauenfelder, H., et al. (1991) Science 254, 1598-1603], the slow annealing of the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pathway. The data suggest that the core of the protein undergoes continued structural rearrangements affecting the rigidity of the protein environment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.

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↗

PubMed1993

Detection of intermediate protein conformations by room temperature tryptophan phosphorescence spectroscopy during denaturation of Escherichia coli alkaline phosphatase.

The reversible denaturation of Escherichia coli alkaline phosphatase (AP) was followed by monitoring changes in enzymatic activity as well as by measurements of the time-resolved room temperature phosphorescence from Trp 109. It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) inactive AP by different mechanisms as reflected by differences in the time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate. Time-resolved tryptophan phosphorescence is exquisitely sensitive to changes in the local environment of the emitting residue, unlike the steady state phosphorescence intensity which is a composite of both the lifetime and concentration of the emitting protein species. The results show that while inactivation in EDTA proceeds by loss of the zinc ion as expected, denaturation in acid or GdnHCl produces a heterogeneous population of AP molecules, detected by a distribution analysis of the phosphorescence lifetime, which may reflect multiple pathways to the final unfolded state. Time-resolved phosphorescence also demonstrates the existence of an enzymatically active but structurally less rigid intermediate state during unfolding. As the rigidity decreases, the susceptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration. The experiments provide new insight into the mechanism of denaturation of AP and demonstrate the sensitivity of time-resolved room temperature phosphorescence to the structural details of intermediate states produced during unfolding of proteins.

Alkaline Phosphatase↗

PubMed1992

Time-resolved circularly polarized protein phosphorescence.

The existence of circular polarization in room-temperature protein phosphorescence is demonstrated, and time-resolved circularly polarized phosphorescence (TR-CPP) is used to characterize unique tryptophan environments in multitryptophan proteins. Circularly polarized luminescence studies provide information regarding the excited state chirality of a lumiphore which can be used to extract sensitive structural information. It is shown by time resolving the circular polarization that it is possible to correlate the excited state chirality with unique decay components in a multiexponential phosphorescence decay profile. The present study presents a concurrent analysis of room-temperature time-resolved phosphorescence and TR-CPP of bacterial glucose-6-phosphate dehydrogenase as well as those of horse liver alcohol dehydrogenase. Only one of the two tryptophan residues per subunit of dimeric alcohol dehydrogenase is believed to phosphorescence, while the dimeric glucose-6-phosphate dehydrogenase has eight tryptophan residues per subunit and shows a corresponding complexity in its phosphorescence decay profile. The anisotropy factor [g(em) = delta I/(Itotal/2); delta I = Ileft circular-Iright circular] for alcohol dehydrogenase is time independent, suggesting a unique excited state chirality. The phosphorescence decay of glucose-6-phosphate dehydrogenase can be well fitted with four exponential terms of 4, 23, 76, and 142 msec, and the TR-CPP of this enzyme shows a strong time dependence that can be resolved into four individual time-independent anisotropy factors of -4.0, -2.1, +6.5, and +6.9 (x10(-3)), each respectively associated with one of the four lifetime components. These results demonstrate how the use of TR-CPP can facilitate the study of proteins with multiple lumiphores.

Alcohol Dehydrogenase↗

PubMed1992

Consideration of dipole orientation angles yields accurate rate equations for energy transfer in the rapid diffusion limit.

Dipole-dipole energy transfer between suitable donor and acceptor chromophores is an important luminescence quenching mechanism and has been shown to be useful for distance determination at the molecular level. In the rapid diffusion limit, where the excited-state lifetime of the donor is long enough to allow the donor and acceptor to diffuse many times their average separation before deexcitation, it is usually assumed that the relative dipolar orientation is completely averaged due to rotational Brownian motion. Under this simplifying assumption, analytical expressions have been derived earlier for the energy transfer rate between donor and acceptor characterized by different geometries. Most such expressions, however, are only approximate because complete angular averaging is permitted only in a geometry that possesses spherical symmetry surrounding each chromophore. In this paper analytical expressions that correctly account for incomplete angle averaging due to steric hindrance are presented for several geometries. Each of the equations reveals a dependence of the energy transfer rate on chromophore orientation. It is shown that correctly accounting for this effect can lead to improvements in estimates of the distance of closest approach from measured quenching rates based on energy transfer experiments.

Diffusion↗

PubMed1991

Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit.

Quenching of the room-temperature phosphorescence of Escherichia coli alkaline phosphatase by several freely diffusing molecules was studied, each of whose absorption spectrum overlaps the long-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced Förster energy transfer. The presence of additional nonresonance transfer mechanisms was also detected, from a lack of linear dependence of quenching rate on spectral overlap. The quenching agents used were the dye molecules methyl red, methyl orange, and 2-[(4-hydroxyphenyl)azo]benzoic acid, as well as the embedded heme groups of myoglobin, metmyoglobin, and the reduced and oxidized forms of cytochrome c. Quenching was found to be greatly diminished upon reduction of each acceptor, indicating that electron transfer occurs efficiently from the excited tryptophan to the oxidized form of the acceptors. The elimination of this electron transfer in the reduced form affords the opportunity to separately measure the Förster transfer rates for the heme proteins. When the transfer rate constant thus measured for myoglobin is applied to a model where both donor and acceptor proteins are taken to be spherical with both tryptophan and the heme group placed off center (a model whose quenching rate equation is newly presented here), the depth of the phosphorescent tryptophan beneath the surface of alkaline phosphatase is found to be 16 A. This value is close to the depth of tryptophan 109 (which is known to be the phosphorescent residue in alkaline phosphatase), showing that with properly chosen probes this technique is indeed valuable for distance determinations in protein structure studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗