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P T Williamson

Publications and source records attributed to P T Williamson.

8 recordsLinked to original sources
PubMed2001

Dynamics and orientation of N+(CD3)3-bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor.

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N(+)(CD(3))(3)-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42 degrees in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

Acetylcholine↗
PubMed2000

Expression and purification of recombinant neurotensin in Escherichia coli.

An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques.

Chromatography, High Pressure Liquid↗
PubMed1999

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1998

Probing the agonist binding pocket in the nicotinic acetylcholine receptor: a high-resolution solid-state NMR approach.

Acetylcholine, the agonist for the nicotinic acetylcholine receptor, has been observed directly when bound specifically to its binding site in the fully functional receptor-enriched membranes from Torpedo nobiliana. High-resolution solid-state, magic angle spinning 13C NMR methods have been used to observe selectively N+(13CH3)3 acetylcholine bound in as few as 20 nmol of receptor binding sites, against a background of natural abundance membrane resonances and excess acetylcholine in free solution. The specificity of the binding has been demonstrated to be pharmacologically significant through the use of the competitive inhibitor alpha bungarotoxin which selectively displaces and prevents binding of acetylcholine to the membrane-bound receptor. The chemical shift assigned to N+(13CH3)3 acetylcholine in solution and crystalline solid is 53.9 +/- 0.04 ppm, and it changes by 1.6 ppm (p < 0.05) for agonist when bound specifically in the receptor binding site. Through the use of computer simulations of chemical shifts carried out on acetylcholine bound to the acetylcholinesterase, we propose that the cause for this change is the presence of aromatic side chains lining the receptor binding site. It is suggested that the binding of acetylcholine to the nicotinic acetylcholine receptor is mediated primarily through the interaction of the quaternary ammonium group of the acetylcholine with the pi bonded systems in the aromatic side chains. Longitudinal relaxation time measurements show that the residency time for the acetylcholine observed in DDCP experiments is long (> 200 ms) with respect to the longitudinal relaxation time of other assignable resonances within the spectrum from the lipid and protein and confirms that the acetylcholine is protein-associated, and not free in solution or nonspecifically bound.

Acetylcholine↗
PubMed1997

Macroscopic orientation of natural and model membranes for structural studies.

One approach for obtaining high-resolution structural and functional information for biomembranes and their proteins is by static solid-state NMR of oriented systems. Here, a general procedure to align fully functional biological membranes containing large membrane proteins (Mr >30,000) is described. The method, based on the isopotential spin-dry ultracentrifugation technique, relies on the centrifugation of membrane fragments onto a support with simultaneous, or subsequent, partial evaporation of the solvent which aids alignment. The quality of orientation, as shown by the mosaic spread of the samples, was monitored by static solid-state 31P NMR for the phospholipids and by 2H NMR for a deuterated retinal in bovine rhodopsin. The generality of this method is demonstrated with three different membranes containing bovine rhodopsin in reconstituted bilayers, natural membranes with the red cell anion exchange transport protein in erythrocytes, band 3, and the nicotinic acetylcholine receptor.

Animals↗