Craig J. Morton

Solution Structure of the Link Module: A Hyaluronan-Binding Domain Involved in Extracellular Matrix Stability and Cell Migration

Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.

The effects of guanidine hydrochloride on the 'random coil' conformations and NMR chemical shifts of the peptide series GGXGG.

The effects of the commonly used denaturant guanidine hydrochloride(GuHCl) on the random coil conformations and NMR chemical shifts of theproteogenic amino acids have been characterized using the peptide seriesAc-Gly-Gly-X-Gly-Gly-NH2. The φ angle-sensitive couplingconstants, ROESY cross peak intensities and proline cis–trans isomerratios of a representative subset of these peptides are unaffected by GuHCl,which suggests that the denaturant does not significantly perturb intrinsicbackbone conformational preferences. A set of3JHNHα values is presented which agreewell with predictions of recently developed models of the random coil. Wehave also measured the chemical shifts of all 20 proteogenic amino acids inthese peptides over a range of GuHCl concentrations. The shifts exhibit alinear dependence on denaturant concentration and we report here correctionfactors for the calculation of ‘random coil’ 1H chemicalshifts at any arbitrary denaturant concentration. Studies of arepresentative subset of peptides indicate that 13C and15N chemical shifts are also perturbed by the denaturant.These results should facilitate the application of chemical shift-basedanalytical techniques to the study of polypeptides in solution with GuHCl.The effects of the denaturant on the quality of NMR spectra and on chemicalshift referencing are also addressed.

Solid-state NMR structure determination of melittin in a lipid environment.

Solid-state (13)C NMR spectroscopy was used to investigate the three-dimensional structure of melittin as lyophilized powder and in ditetradecylphosphatidylcholine (DTPC) membranes. The distance between specifically labeled carbons in analogs [1-(13)C]Gly3-[2-(13)C]Ala4, [1-(13)C]Gly3-[2-(13)C]Leu6, [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 was measured by rotational resonance. As expected, the internuclear distances measured in [1-(13)C]Gly3-[2-(13)C]Ala4 and [1-(13)C]Gly3-[2-(13)C]Leu6 were consistent with alpha-helical structure in the N-terminus irrespective of environment. The internuclear distances measured in [1-(13)C]Leu13-[2-(13)C]Ala15, [2-(13)C]Leu13-[1-(13)C]Ala15, and [1-(13)C]Leu13-[2-(13)C]Leu16 revealed, via molecular modeling, some dependence upon environment for conformation in the region of the bend in helical structure induced by Pro14. A slightly larger interhelical angle between the N- and C-terminal helices was indicated for peptide in dry or hydrated gel state DTPC (139 degrees -145 degrees ) than in lyophilized powder (121 degrees -139 degrees ) or crystals (129 degrees ). The angle, however, is not as great as deduced for melittin in aligned bilayers of DTPC in the liquid-crystalline state (approximately 160 degrees ). The study illustrates the utility of rotational resonance in determining local structure within peptide-lipid complexes.

The speciation of gold and copper cyanide complexes on ion-exchange resins containing different functional groups

Abstract Despite the work of many researchers on the use of ion exchange technology for the recovery of gold from cyanide leached slurries, very little work has considered the effect that the chemical structure and hydrophilicity of the functional group may have on the speciation of the sorbed metal cyanide species. The present study investigated the properties of five resins that have the same type of resin matrix but contain a different aliphatic amino functional group. The tested resins include a variety of predominantly weak base resins that contain a small amount of strong base groups. These types of resin are similar to those that are currently being used in resin-in-pulp processes in the former Soviet Union for the recovery of gold (AM-2B). This study used CP/MAS 13 C-NMR to determine the chemical structure of the functional group on each synthesised resin. Raman spectroscopy was used in conjunction with FTIR spectroscopy to determine the speciation of copper(I)–cyanide and gold(I)–cyanide on each resin studied. Despite the equilibrium distribution of copper cyanide species in solution it was established that [Cu(CN) 3 ] 2− predominantly loaded onto all resins studied. However, for resins of a low ionic density the sorption of [Cu(CN) 2 ] − was also observed. Raman spectroscopy showed that gold cyanide loads onto each resin as the linear [Au(CN) 2 ] − complex and that no change in speciation was observed in highly saline solutions. The observed phenomena have been used to successfully explain the selective sorption properties of each resin in non-saline and highly saline solutions.

Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

Solid-state NMR conformational studies of a melittin-inhibitor complex.

Abstract. Melittin is a cytolytic peptide whose biological activity is lost upon binding to a six-residue peptide, Ac-IVIFDC-NH2, with which it forms a highly insoluble complex. As a result, the structural analysis of the interaction between the two peptides is difficult. Solid-state NMR spectroscopy was used to study the interaction between melittin and the peptide inhibitor. Location of the binding site in the melittin-inhibitor complex was determined using lanthanide ions, which quench NMR resonances from molecular sites that are in close proximity to the unique ion binding site. Our results indicated that the inhibitor binding site in melittin is near Leu13, Leu16 and Ile17, but not near Leu6 or Val8. On the basis of these data we propose that the inhibitor binds to melittin in the vicinity of Ala15 to Trp19 and prevents insertion of melittin into cell membranes by disrupting the helical structure. Supporting evidence for this model was produced by determining the distance, using rotational resonance NMR, between the [1-13C] of Leu13 in melittin and the [3-13C] of Phe4 in the inhibitor.

NMR STUDIES OF THE SOLUTION PROPERTIES OF RECOMBINANT MURINE INTERLEUKIN-6

The effects of solvent, pH and temperature on the 1H-NMR spectra of recombinant murine interleukin-6 (IL-6) are described. Assignments made from two-dimensional homonuclear spectra are presented for resonances of the fifteen aromatic amino-acid side chains. A time-dependent loss of intensity was observed for all resonances in the spectrum of IL-6, probably as a result of aggregation. This aggregation is markedly temperature-dependent. The pKa values of the four histidine residues in murine IL-6 has been measured; one has a value of 5.5, approx. one pH unit less than the value exhibited by the other three. Analysis of the NOESY spectra has allowed a preliminary characterisation of the nature of interactions among the aromatic side chains within the protein fold. 1H and 15N resonances of residues Thr-4 to Val-21 are assigned from three-dimensional 1H-15N correlated spectroscopy, and evidence is presented for these residues comprising a mobile N-terminal tail with little ordered structure. An N-terminal mutant lacking the first 22 residues of the murine IL-6 sequence and known to possess full biological activity was also examined and shown to have essentially retained the tertiary fold of the native molecule.

Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A

Biotinylated gramicidins are an important component of the AMBRI® “ion channel switch™” biosensor. These gramicidin A (gA) analogues have a biotin attached to the C-terminus of gA via a number of aminocaproyl linker groups (X). The structure of gA5XB has been determined in deuterated sodium dodecyl sulfate micelles and is similar to native gA and other modified gA analogues. The biotin and aminocaproyl groups were mobile and located in the aqueous phase and when avidin was added, NMR and MS studies showed that gA5XB bound more effectively to avidin than gA2XB. The length and flexibility of the linker appears to be important for biotin–avidin binding and, in the AMBRI® biosensor, gA5XB is a more effective gated ion channel than gA2XB. The conformation and dynamics of the aminocaproyl linker groups were investigated using 2H solid-state NMR. Deuterated aminocaproyl linkers were coupled to gA and incorporated into oriented bilayers in order to analyse the order and dynamics of the aminocaproyl linker. The small 2H splittings and the T1 relaxation times indicated that the aminocaproyl linker is undergoing fast rotation in phospholipid bilayers. Native d4-gA as well as d4-gA2XB, where the ethanolamine has been deuterated, were also incorporated into oriented bilayers. Solid-state 2H NMR data showed that the addition of the linker group restricted the mobility of the ethanolamine. However, these modifications to the C-terminus of gA did not interfere with ion channel function and clarify how the biotinylated gA analogues perform in the lipid bilayer as part of the AMBRI® biosensor.

Cholesterol-dependent cytolysins: from water-soluble state to membrane pore

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.