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Harry B Gray

Publications and source records attributed to Harry B Gray.

64 records · Page 4Linked to original sources
PubMed2003

Trapping of peptide-based surrogates in an artificially created channel of cytochrome c peroxidase.

As recently described, the deliberate removal of the proposed electron transfer pathway from cytochrome c peroxidase resulted in the formation of an extended ligand-binding channel. The engineered channel formed a template for the removed peptide segment, suggesting that synthetic surrogates might be introduced to replace the native electron transfer pathway. This approach could be united with the recent development of sensitizer-linked substrates to initiate and study electron transfer, allowing access to unresolved issues about redox mechanism of the enzyme. Here, we present the design, synthesis, and screening of a peptide library containing natural and unnatural amino acids to identify the structural determinants for binding this channel mutant. Only one peptide, (benzimidazole-propionic acid)-Gly-Ala-Ala, appeared to interact, and gave evidence for both reversible and kinetically trapped binding, suggesting multiple conformations for the channel protein. Notably, this peptide was the most analogous to the removed electron transfer sequence, supporting the use of a cavity-template strategy for design of specific sensitizer-linked substrates as replacements for the native electron transfer pathway.

Cytochrome-c Peroxidase↗
PubMed2002

Structural features of the cytochrome C molten globule revealed by fluorescence energy transfer kinetics.

Nonnative states of proteins are involved in a variety of cellular processes, including translocation of proteins across membranes and formation of amyloid fibrils. Probes that report on the structural heterogeneity of a polypeptide ensemble could resolve ambiguities in the classification of these states. Employing fluorescence energy transfer kinetics, we have shown that added anions shift the equilibrium between the compact and extended polypeptide structures that are present during refolding of Saccaromyces cerevisiae iso-1 cytochrome c. Specifically, at high salt concentrations (>/=700 mM), all of the polypeptides are compact with a mean C-terminal fluorophore-heme separation quite close to that in the native protein (25 A).

Circular Dichroism↗
PubMed2002

Structural features of cytochrome c' folding intermediates revealed by fluorescence energy-transfer kinetics.

We employed fluorescence energy-transfer probes to investigate the polypeptide dynamics accompanying cytochrome c' folding. Analysis of fluorescence energy-transfer kinetics from wild-type Trp-72 or Trp-32 in a crystallographically characterized (1.78 A) Q1A/F32W/W72F mutant shows that there is structural heterogeneity in denatured cytochrome c'. Even at guanidine hydrochloride concentrations well beyond the unfolding transition, a substantial fraction of the polypeptides ( approximately 50%) adopts compact conformations (tryptophan-to-heme distance, approximately 25 A) in both pseudo-wild-type (Q1A) and mutant proteins. A burst phase (< or =5 ms) is revealed when stopped flow-triggered refolding is probed by tryptophan intensity: measurements on the Q1A protein show that approximately 75% of the Trp-72 fluorescence (83% for Trp-32) is quenched within the mixing deadtime, suggesting that most of the polypeptides have collapsed.

Amino Acids↗
PubMed2002

Fluorescent probes for cytochrome p450 structural characterization and inhibitor screening.

We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Förster energy transfer upon binding (Kd = 0.83 muM) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (Kd approximately 0.02 muM) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 A resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the B', F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors.

Adamantane↗
PubMed2002

The cytochrome c folding landscape revealed by electron-transfer kinetics.

We have investigated the folding energy landscape of cytochrome c by exploiting the widely different electron-transfer (ET) reactivities of buried and exposed Zn(II)-substituted hemes. An electronically excited Zn-porphyrin in guanidine hydrochloride denatured Zn-substituted cytochrome c (Zn-cyt c) reduces ruthenium(III) hexaammine about ten times faster than when embedded in the fully folded protein. Measurements of ET kinetics during Zn-cyt c folding reveal a burst intermediate in which one-third of the ensemble has a protected Zn-porphyrin and slow ET kinetics; the remaining fraction exhibits fast ET characteristic of a solvent-exposed redox cofactor. The ET data show that, under solvent conditions favoring the folded protein, collapsed non-native structures are not substantially more stable than extended conformations, and that the two populations interchange rapidly. Most of the folding free energy, then, is released when compact structures evolve into the native fold.

Cytochrome c Group↗
PubMed2002

Using deeply trapped intermediates to map the cytochrome c folding landscape.

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome c. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal-ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome c.

Animals↗
PubMed2002

Mapping the cytochrome C folding landscape.

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S.cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a time scale regulated by heme ligation events ( approximately 300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.

Cytochrome c Group↗
PubMed2002

An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins.

In azurins and other blue copper proteins with relatively low reduction potentials (E(0) [Cu(II)/Cu(I)]<400 mV vs. normal hydrogen electrode), the folded polypeptide framework constrains both copper(II) and copper(I) in such a way as to tune the reduction potentials to values that differ greatly from those for most copper complexes. Largely conserved networks of hydrogen bonds organize and lock the rest of the folded protein structure to a loop that contains three of the ligands to copper. Changes in hydrogen bonds that allow copper(I) to revert more closely to its preferred geometry [relative to the copper(II) geometry] accordingly lead to an increase in E(0). This paper reports mutations in the ligand loop of amicyanin from P. denitrificans that relax the constraints on ligation for copper(I) and significantly raise E(0) for these mutants (for example 415+/-4 mV) relative to that of the native amicyanin (265+/-4 mV). These mutations also shift the pK(a) of a ligand histidine to below 5 relative to 7.0 in the wild type.

Bacterial Proteins↗
PubMedDate not supplied

Amphiphilic corroles bind tightly to human serum albumin.

Amphiphilic 2,17-bis-sulfonato-5,10,15(trispentafluorophenyl)corrole (2) and its Ga and Mn complexes (2-Ga and 2-Mn) form tightly bound noncovalent conjugates with human serum albumin (HSA). Protein-induced changes in the electronic absorption, emission, and circular dichroism spectra of these corroles, as well as results obtained from HPLC profiles of the conjugates and selective fluorescence quenching of the single HSA tryptophan, are interpreted in terms of multiple corrole:HSA binding sites. High-affinity binding sites, close to the unique tryptophan, are fully occupied at very low concentrations. At biologically relevant HSA concentrations (2-3 orders of magnitude larger than those employed in our studies), all corroles (2, 2-Ga, and 2-Mn) may be considered as fully conjugated.

Binding Sites↗