Gary J. Sharman

Prion protein fragments spanning helix 1 and both strands of β sheet (residues 125–170) show evidence for predominantly helical propensity by CD and NMR

BACKGROUND Transmissible spongiform encephalopathies are a group of neurodegenerative disorders of man and animals that are believed to be caused by an alpha-helical to beta-sheet conformational change in the prion protein, PrP. Recently determined NMR structures of recombinant PrP (residues 121-231 and 90-231) have identified a short two-stranded anti-parallel beta sheet in the normal cellular form of the protein (PrPC). This beta sheet has been suggested to be involved in seeding the conformational transition to the disease-associated form (PrPSc) via a partially unfolded intermediate state. RESULTS We describe CD and NMR studies of three peptides (125-170, 142-170 and 156-170) that span the beta-sheet and helix 1 region of PrP, forming a large part of the putative PrPSc-PrPC binding site that has been proposed to be important for self-seeding replication of PrPSc. The data suggest that all three peptides in water have predominantly helical propensities, which are enhanced in aqueous methanol (as judged by deviations from random-coil Halpha chemical shifts and 3JHalpha-NH values). Although the helical propensity is most marked in the region corresponding to helix 1 (144-154), it is also apparent for residues spanning the two beta-strand sequences. CONCLUSIONS We have attempted to model the conformational properties of a partially unfolded state of PrP using peptide fragments spanning the region 125-170. We find no evidence in the sequence for any intrinsic conformational preference for the formation of extended beta-like structure that might be involved in promoting the PrPC-PrPSc conformational transition.

Dissecting the effects of cooperativity on the stabilisation of a de novo designed three stranded anti-parallel β-sheet

Cooperative effects between two sets of weak interactions at different hydrogen bonded interfaces are shown to stabilise a de novo designed three-stranded anti-parallel β-sheet: NMR and CD (circular dichroism) spectroscopy have been used to compare and contrast the stability of the β-sheet (residues 1–24) and the isolated C-terminal β-hairpin (residues 9–24).

Enthalpic (electrostatic) contribution to the chelate effect: a correlation between ligand binding constant and a specific hydrogen bond strength in complexes of glycopeptide antibiotics with cell wall analogues

The 1H NMR chemical shift of amide protons in the binding pocket of glycopeptide antibiotics has been used to monitor the interaction of these amide protons with the carboxylate group of cell wall analogues and related ligands. A good correlation is observed between overall ligand binding energy (ΔG°) and amide NH chemical shift. We conclude that the strength of the electrostatic interaction of the carboxylate group, which is crucial to recognition and binding by the antibiotics, is cooperatively enhanced by adjacent functional groups on the same ligand template. Hydrogen bonding and burial of hydrocarbon in adjacent sites produce an enhancement of electrostatic binding of the carboxylate group. The data provide experimental evidence for an enthalpic contribution to the chelate effect that is distinct from, and works in addition to, the classic entropic chelate effect. The correlation between amide NH chemical shift and overall binding energy has been used to show binding affinity for eremomycin and chloroeremomcin by di-N-Ac-Lys-D-Ala-D-Lac (Lac = lactate), which is a cell wall analogue of bacteria which exhibit vancomycin resistance. Binding constants for this ligand have also been determined by UV difference spectrophotometry (70 dm3 mol–1 and 240 dm3 mol–1 respectively).

Common factors in the mode of action of vancomycin group antibiotics active against resistant bacteria

It is shown that a semisynthetic glycopeptide, with activity against vancomycin resistant bacteria, possesses (as do other glycopeptides active in this way) features which promote its dimerisation and membrane anchoring.

Use of model cell membranes to demonstrate templated binding of vancomycin group antibiotics

In this paper we demonstrate the importance of binding geometry and dimerisation at the surface of model cell membranes in the mode of action of the clinically important glycopeptide antibiotics. This has been achieved through the use of model cell membranes (micelles and vesicles) to which cell wall analogues are anchored via a hydrophobic decanoyl chain. A number of –D-Ala-terminating cell wall analogues, ranging from two to six residues in length, have been used. Dipeptide, pentapeptide and hexapeptide display enhanced binding to the antibiotic at the model cell surface, but tripeptide and tetrapeptide do not. The possible implications of the observed binding geometries for bacterial systems are discussed.

Binding of a vancomycin group antibiotic to a cell wall analogue from vancomycin-resistant bacteria

Glycopeptide antibiotics bind to bacterial cell wall peptide analogues terminating in -L-Lys-D-Ala-D-Lac in a similar manner to that of cell-wall analogues terminating in -LLys-D-Ala-D-Ala.

Development of a selective TOCSY experiment and its use in analysis of a mixture of related compounds

A selective TOCSY experiment based on the recently described DPFGSE selective excitation sequence has been developed: it employs gradient purging pulses which result in excellent lineshapes free of antiphase dispersive components, and has proven useful in the identification of a number of impurities in a pharmaceutical intermediate.

Evidence for β-sheet conformation in vesicle-bound peptides derived from the transmembrane bacterial flagellar motor protein MotB from Rhodobacter sphaeroides

Circular dichroism experiments show that a 28 residue transmembrane peptide derived from the R. sphaeroides bacterial flagellar motor protein MotB adopts predominantly β-sheet conformation when bound within phosphatidylcholine vesicles. A peptide with a mutation at Asp32, which has been shown to destroy proton conductance, is also shown to insert into the model membrane in predominantly β-sheet conformation, suggesting that it is not the gross structural features of the transmembrane region that are disrupted by this mutation but perhaps only the electrostatic properties of the pore. A tentative structure for a MotB pore is proposed which consists of an eight stranded β-barrel.

Enhancement of Electrostatic Binding Through Cooperative Interactions: Enthalpy/Entropy Compensation and Peptide—Peptide Recognition

In recent work we have established a fundamental relationship between the cost in entropy of an association between two entities A and B, involving one- and two-point interactions in the gas phase, or in non-polar solvents, to give a complex A,B, as a function of the exothermicity of the interaction between them (1). While there is a limit to the adverse entropy of a bimolecular association in terms of the loss of translational and overall rotational freedom (ca 50 to 60 kJ/mol for TΔS° in solution at 298 K) (2), the exothermicity of an interaction can increase far beyond the exothermicity at which the limiting cost in entropy is approached. We have proposed, and subsequently justified on the basis of a large body of experimental data from many laboratories (for example, see Figure 1A), that the enthalpic benefit versus entropie cost of associations of the type stipulated above have the general form shown in Figure 1B (1). We emphasise the approximate form of the curve; the precise entropie cost of an association is dependent on many variables such as mass, density of vibrational states and the shape of the potential energy well in which the associated species lies. In complex systems where the possibility arises that internal rotations are also restricted within the associating species, then the limiting entropie cost will be higher with the curve displaced to the right. The loss of entropy illustrated by Figures 1A and 1B reflects only loss of translational and overall rotational freedom upon association. The data clearly show that associations with small exothermicities can result in remarkably small adverse entropy changes.