Biomedical subjects

Mike P Williamson

Publications and source records attributed to Mike P Williamson.

8 recordsLinked to original sources

PubMed2006

The solution structure of the PufX polypeptide from Rhodobacter sphaeroides.

PufX organizes the photosynthetic reaction centre-light harvesting complex 1 (RC-LH1) core complex of Rhodobacter sphaeroides and facilitates quinol/quinone exchange between the RC and cytochrome bc(1) complexes. The structure of PufX in organic solvent reveals two hydrophobic helices flanked by unstructured termini and connected by a helical bend. The proposed location of basic residues and tryptophans at the membrane interface orients the C-terminal helix along the membrane normal, with the GXXXG motifs in positions unsuitable as direct drivers of dimerisation of the RC-LH1 complex. The N-terminal helix is predicted to extend approximately 40 Anggstrom along the membrane interface.

Amino Acid Sequence↗

PubMed2006

Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy.

BACKGROUND: The green tea flavonoid, epigallocatechin gallate (EGCG), has been proposed to have an anti-HIV-1 effect by preventing the binding of HIV-1 glycoprotein (gp) 120 to the CD4 molecule on T cells. OBJECTIVE: To demonstrate that EGCG binds to the CD4 molecule at the gp120 attachment site and inhibits gp120 binding at physiologically relevant levels, thus establishing EGCG as a potential therapeutic treatment for HIV-1 infection. METHODS: Nuclear magnetic resonance spectroscopy was used to examine the binding of EGCG and control, (-)-catechin, to CD4-IgG2 (PRO 542). Gp120 binding to human CD4+ T cells was analyzed by flow cytometry. RESULTS: Addition of CD4 to EGCG produced a linear decrease in nuclear magnetic resonance signal intensity from EGCG but not from the control, (-)-catechin. In saturation transfer difference experiments, addition of 5.8 micromol/L CD4 to 310 micromol/L EGCG produced strong saturation at the aromatic rings of EGCG, but identical concentrations of (-)-catechin produced much smaller effects, implying EGCG/CD4 binding strong enough to reduce gp120/CD4 binding substantially. Molecular modeling studies suggested a binding site for EGCG in the D1 domain of CD4, the pocket that binds gp120. Physiologically relevant concentrations of EGCG (0.2 micromol/L) inhibited binding of gp120 to isolated human CD4+ T cells. CONCLUSION: We have demonstrated clear evidence of high-affinity binding of EGCG to the CD4 molecule with a Kd of approximately 10 nmol/L and inhibition of gp120 binding to human CD4+ T cells. CLINICAL IMPLICATIONS: Epigallocatechin gallate has potential use as adjunctive therapy in HIV-1 infection.

Binding Sites↗

PubMed2006

Binding of amyloid beta-peptide to ganglioside micelles is dependent on histidine-13.

Amyloid beta-peptide (Abeta) is a major component of plaques in Alzheimer's disease, and formation of senile plaques has been suggested to originate from regions of neuronal membrane rich in gangliosides. Here we demonstrate using NMR on 15N-labelled Abeta-(1-40) and Abeta-(1-42) that the interaction with ganglioside G(M1) micelles is localized to the N-terminal region of the peptide, particularly residues His13 to Leu17, which become more helical when bound. The key interaction is with His13, which undergoes a G(M1)-specific conformational change. The sialic acid residue of the ganglioside headgroup is important for determining the nature of the conformational change. The isolated pentasaccharide headgroup of G(M1) is not bound, suggesting the need for a polyanionic surface. Binding to heparin confirms this suggestion, since binding is of similar affinity but does not produce the same conformational changes in the peptide. A comparison of Abeta-(1-40) and Abeta-(1-42) indicates that binding to G(M1) micelles is not related to oligomerization, which occurs at the C-terminal end. These results imply that binding to ganglioside micelles causes a transition from random coil to alpha-helix in the N-terminal region, leaving the C-terminal region unstructured.

Amyloid beta-Peptides↗

PubMed2006

Noncovalent cross-linking of casein by epigallocatechin gallate characterized by single molecule force microscopy.

Interaction of the tea polyphenol epigallocatechin gallate (EGCG) with beta-casein in milk affects the taste of tea and also affects the stability of the tea and the antioxidant ability of the EGCG. In addition, interaction of polyphenols with the chemically similar salivary proline-rich proteins is largely responsible for the astringency of tea and red wine. With the use of single molecule force microscopy, we demonstrate that the interaction of EGCG with a single casein molecule is multivalent and leads to reduction in the persistence length of casein as calculated using the wormlike chain model and a reduction in its radius of gyration. The extra force required to stretch casein in the presence of EGCG is largely entropic, suggesting that multivalent hydrophobic interactions cause a compaction of the casein micelle.

Caseins↗

PubMed2005

Structure of a mannan-specific family 35 carbohydrate-binding module: evidence for significant conformational changes upon ligand binding.

Enzymes that digest plant cell wall polysaccharides generally contain non-catalytic, carbohydrate-binding modules (CBMs) that function by attaching the enzyme to the substrate, potentiating catalytic activity. Here, we present the first structure of a family 35 CBM, derived from the Cellvibrio japonicus beta-1,4-mannanase Man5C. The NMR structure has been determined for both the free protein and the protein bound to mannopentaose. The data show that the protein displays a typical beta-jelly-roll fold. Ligand binding is not located on the concave surface of the protein, as occurs in many CBMs that display the jelly-roll fold, but is formed by the loops that link the two beta-sheets of the protein, similar to family 6 CBMs. In contrast to the majority of CBMs, which are generally rigid proteins, CBM35 undergoes significant conformational change upon ligand binding. The curvature of the binding site and the narrow binding cleft are likely to be the main determinants of binding specificity. The predicted solvent exposure of O6 at several subsites provides an explanation for the observed accommodation of decorated mannans. Two of the key aromatic residues in Man5C-CBM35 that interact with mannopentaose are conserved in mannanase-derived CBM35s, which will guide specificity predictions based on the primary sequence of proteins in this CBM family.

Amino Acid Sequence↗

PubMed2005

An experimental investigation of conformational fluctuations in proteins G and L.

The B1 domains of streptococcal proteins G and L are structurally similar, but they have different sequences and they fold differently. We have measured their NMR spectra at variable temperature using a range of concentrations of denaturant. Many residues have curved amide proton temperature dependence, indicating that they significantly populate alternative, locally unfolded conformations. The results, therefore, provide a view of the locations of low-lying, locally unfolded conformations. They indicate approximately 4-6 local minima for each protein, all within ca. 2.5 kcal/mol of the native state, implying a locally rough energy landscape. Comparison with folding data for these proteins shows that folding involves most molecules traversing a similar path, once a transition state containing a beta hairpin has been formed, thereby defining a well-populated pathway down the folding funnel. The hairpin that directs the folding pathway differs for the two proteins and remains the most stable part of the folded protein.

Bacterial Proteins↗

PubMed2004

Copper-mediated LDL oxidation by homocysteine and related compounds depends largely on copper ligation.

Oxidation of low-density lipoprotein (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Elevated plasma homocysteine is an accepted risk factor for atherosclerosis, and may act through LDL oxidation, although this is controversial. In this study, homocysteine at physiological concentrations is shown to act as a pro-oxidant for three stages of copper-mediated LDL oxidation (initiation, conjugated diene formation and aldehyde formation), whereas at high concentration, it acts as an antioxidant. The affinity for copper of homocysteine and related copper ligands homocysteine, cystathionine and djenkolate was measured, showing that at high concentrations (100 microM) under our assay conditions, they bind essentially all of the copper present. This is used to rationalise the behaviour of these ligands, which stimulate LDL oxidation at low concentration but generally inhibit it at high concentration. Albumin strongly reduced the effect of homocystine on lag time for LDL oxidation, suggesting that the effects of homocystine are due to copper binding. In contrast, copper binding does not fully explain the pro-oxidant behaviour of low concentrations of homocysteine towards LDL, which appears in part at least to be due to stimulation of free radical production. The likely role of homocysteine in LDL oxidation in vivo is discussed in the light of these results.

Copper↗

PubMedDate not supplied

Molecular model for astringency produced by polyphenol/protein interactions.

Polyphenols are responsible for the astringency of many beverages and foods. This is thought to be caused by the interaction of polyphenols with basic salivary proline-rich proteins (PRPs). It is widely assumed that the molecular origin of astringency is the precipitation of PRPs following polyphenol binding and the consequent change to the mucous layer in the mouth. Here, we use a variety of biophysical techniques on a simple model system, the binding of beta-casein to epigallocatechin gallate (EGCG). We show that at low EGCG ratios, small soluble polydisperse particles are formed, which aggregate to form larger particles as EGCG is added. There is an initial compaction of the protein as it binds to the polyphenol, but the particle subsequently increases in size as EGCG is added because of the incorporation of EGCG and then to aggregation and precipitation. These results are shown to be compatible with what is known of astringency in foodstuffs.

Circular Dichroism↗