Biomedical subjects
Publications and source records attributed to G L McLendon.
Synthetic receptor 1 has been found via fluorescence titration to compete effectively with cytochrome c peroxidase for binding cytochrome c (Cc), forming 1:1 Cc:1 complex with a binding constant of (3 +/- 1) x 10(8) M-1, and to disrupt Cc: Apaf-1 complex, a key adduct in apoptosis.
Carbon monoxide binding was studied in a collection of de novo heme proteins derived from combinatorial libraries of sequences designed to fold into 4-helix bundles. The design of the de novo sequences was based on the previously reported "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains are varied extensively.(1) The combinatorial mixture of amino acids included histidine and methionine, which ligate heme iron in natural proteins. However, no attempt was made to explicitly design a heme binding site. Nonetheless, as reported previously, approximately half of the binary code proteins bind heme.(2) This collection of novel heme proteins provides a unique opportunity for an unbiased assessment of the functional potentialities of heme proteins that have not been prejudiced either by explicit design or by evolutionary selection. To assess the capabilities of the de novo heme proteins to bind diatomic ligands, we measured the affinity for CO, the kinetics of CO binding and release, and the resonance Raman spectra of the CO complexes for eight de novo heme proteins from two combinatorial libraries. The CO binding affinities for all eight proteins were similar to that of myoglobin, with dissociation constants (K(d)) in the low nanomolar range. The CO association kinetics (k(on)) revealed that the heme environment in all eight of the de novo proteins is partially buried, and the resonance Raman studies indicated that the local environment around the bound CO is devoid of hydrogen-bonding groups. Overall, the CO binding properties of the de novo heme proteins span a narrow range of values near the center of the range observed for diverse families of natural heme proteins. The measured properties of the de novo heme proteins can be considered as a "default" range for CO binding in alpha-helical proteins that have neither been designed to bind heme or CO, nor subjected to genetic selections for heme or CO binding.
Cytochrome c (Cc) binding to apoptosis protease activation factor-1 (Apaf-1) is a critical activation step in the execution phase of apoptosis. Here we report studies that help define the Cc:Apaf-1 binding surface. It is shown that a large number of Cc residues, including residues 7, 25, 39, 62-65, and 72, are involved in the Cc:Apaf-1 interaction. Mutation of residue 72 eliminated Cc activity whereas mutations of residues 7, 25, 39, and 62-65 showed reduced activity in an additive fashion. The implications of this binding model for both recognition and modulation of protein-protein interactions are briefly discussed.
X-ray absorption spectroscopy at the Zn K-edge indicates that the active site of the marine diatom Thalassiosira weissflogii carbonic anhydrase is strikingly similar to that of mammalian alpha-carbonic anhydrase enzymes. The zinc has three histidine ligands and a single water at 1.98 A. This is quite different from the beta-carbonic anhydrases of higher plants in which zinc is coordinated by two cysteine thiolates, one histidine, and a water molecule. The diatom carbonic anhydrase shows no significant sequence similarity with other carbonic anhydrases and may represent an example of convergent evolution at the molecular level.
A general understanding of how cytochromes evolve within a fixed structure to optimize redox potential for specific bioenergetic processes does not exist. Toward this end, a library approach is used to investigate the range and distribution of redox potential which occurs when all sequence space available through mutation at two positions is examined within a fixed structural motif. Random mutation of Phe61 and Phe65 of cytochrome b562 (E. coli), and subsequent examination of a statistically significant sampling of this library, demonstrates that the redox potential can vary over 100 mV (>25% of the known accessible potential in native proteins with axial His-Met ligation) through mutation at these two positions. The redox potential of the wild-type protein occurs at an extremum of the distribution observed, indicating that Phe61 and Phe65 were most likely naturally selected to differentially stabilize the reduced state of the protein. At the other extremum, a compositionally conservative set of mutations (F61I, F65Y) leads to a 100 mV shift in the redox equilibrium toward the oxidized state. NMR analyses indicate that a charge-dipole interaction which results from mutation of phenylalanine to tyrosine at position 65 may be responsible.
Studies of classic zinc-finger peptides over the past 15 years have offered insights into the coupled processes of metal binding and protein folding. Within the past two years, this insight has been used to increase our understanding of the importance of first and second shell contributions (i.e. contributions from direct and indirect metal ligands) to metal binding and protein-folding stability, and led to advances in de novo protein design and protein redesign.
The hdm2 oncoprotein contains a C-terminal domain that binds RNA and has been suggested to bind zinc(II) in an unusual RING finger domain in which Thr 455 was postulated as a ligand. We have reported experiments to test whether this C-terminal cysteine-rich motif is indeed a RING finger domain. We also tested the affinity of the hdm2 C-terminal peptide for metal binding, metal linkage to the folding of the C-terminal peptide, and the peptide's affinity for RNA. Truncation mutants demonstrate that amino acids 425-491 are necessary and sufficient for RNA binding. However, divalent metal ions do not seem to affect the specific RNA recognition. Metal binding studies suggest that hdm2 indeed binds to two molecules of zinc in an intertwined motif similar to the BRCA1 RING finger peptide. However, there is no similarity in overall tertiary structure, nor is there direct sequence homology with other RING fingers. Fluorescence energy transfer studies give a dissociation constant of (0.22 +/- 0.03) microM for cobalt(II) binding to site 1, while K2 for cobalt(II) binding was estimated to be 15 +/- 5 microM from ultraviolet absorbance. Studies of two mutant peptides confirm the assignment of binding residues in hdm2 and suggest that the coordination of Thr 455 previously proposed by sequence alignments is incorrect. Structural studies of hdm2 in the presence and absence of metal indicate only a small amount of secondary structure by circular dichroic spectroscopy. Metal binding did not seem to nucleate folding as in the case of two other RING finger proteins. However, distance measurement from fluorescence energy transfer indicated that the Tyr 489 residue was only approximately 14 A away from the first metal center, suggesting that the hdm2 protein exists in a compact form, at least in the presence of metal ion. In summary, hdm2 binds metal and RNA, but the RNA binding does not seem to occur in a zinc-dependent manner.
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The stereochemical consequences of the metal-ion assisted self-assembly of parallel three-helix peptide bundles are investigated. Chiral induction in the self-assembly of systems containing extensive protein secondary structure is compared with the racemic synthesis of short metallopeptides. Isolation and characterization of the individual stereoisomers of an exchange-inert metalloprotein provide structural insights into analogous exchange-labile systems.
Flash photolysis and pulse radiolysis measurements demonstrate a conformational dependence of electron transfer rates across a 16-mer helical bundle (three-helix metalloprotein) modified with a capping CoIII(bipyridine)3 electron acceptor at the N terminus and a 1-ethyl-1'-ethyl-4,4'- bipyridinium donor at the C terminus. For the CoIII(peptide)3-1-ethyl-1'-ethyl-4,4'-bipyridinium maquettes, the observed transfer is a first order, intramolecular process, independent of peptide concentration or laser pulse energy. In the presence of 6 M urea, the random coil bundle (approximately 0% helicity) has an observed electron transfer rate constant of kobs = 900 +/- 100 s-1. In the presence of 25% trifluoroethanol (TFE), the helicity of the peptide is 80% and the kobs increases to 2000 +/- 200 s-1. Moreover, the increase in the rate constant in TFE is consistent with the observed decrease in donor-acceptor distance in this solvent. Such bifunctional systems provide a class of molecules for testing the effects of conformation on electron transfer in proteins and peptides.
The enzyme p-cresol methylhydroxylase [4-cresol: (acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1] contains two subunits: a cytochrome c (electron transfer) subunit (cytochrome cpc) and a flavin (catalytic) subunit. When these subunits are separated by isoelectric focusing, a stable cytochrome subunit is obtained. Significant differences are observed between the one-dimensional NMR spectra of oxidized cytochrome cpc and of oxidized p-cresol methylhydroxylase. Analysis of the two-dimensional nuclear Overhauser enhancement and exchange spectroscopy (NOESY) spectrum of reduced cytochrome cpc suggests that the axial ligand, Met-50, of the stable subunit reorients by a rotation about the C gamma-S delta bond when cytochrome cpc binds to the flavin subunit. This reorientation must result in a change in bonding at the heme, which is reflected both in the para-magnetically shifted resonances and in the redox potential. p-Cresol methylhydroxylase thereby provides an interesting example of the coupling of subunit interactions to active-site structure and reactivity.
An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1). The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins.
The cyanophycin granule polypeptide, or multi-L-arginyl-poly(L-aspartic acid), from the filamentous cyanobacterium Anabaena cylindrica has been examined by circular dichroism and laser Raman spectroscopy. The polypeptide exhibits defined secondary structure in acidic solution, but not in alkaline medium. Raman spectra show the structure of the insoluble (storage?) form of the polypeptide is similar but not identical to that of the acid soluble form. Analysis of the CD spectra suggests that beta-sheets exist in the acid soluble form, and by inference, in the insoluble cellular material.