Conformational Analysis by Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance spectroscopy is a powerful and versatile spectroscopic technique and novel applications are continually being devised for it. This thesis concerns the application of nmr spectroscopy to the study and definition of both the time dependent and the time independent three dimensional geometries of organic molecules in solution. The purposes of this thesis are: (a) to establish the conceptual background to both nmr spectroscopy and conformational analysis and hence to relate the two fields, (b) to review in varying depth the published results in those areas of chemistry which have been ventured upon in the current research and (c) to present the results and conclus: :ions of the current studies. To these ends, nmr spectroscopy is first briefly reviewed and the areas of major topical interest touched upon. As nmr spectroscopy is a precisely mathematical subject, the theoretical applications later employed are briefly outlined. The thermodynamic considerations relevent to conformational analysis are then reviewed and this is followed by a discussion of the application of nmr spectroscopy to detailed conformational analysis. The current studies involve reviewing two discrete fields of conformational analysis. Firstly, nmr studies of conformations in medium ring compounds are reviewed in depth. The results of a study of syn-3,7-dibromo-cis,cis-cycloocta-1,5-diene are recorded in which a definitive statement of the solution conformation (in chloroform) of this molecule is possible. The consistency of the data from other techniques relating to this conformation is remarked upon and the similarity to the solid state conformation determined (F. B. Wilson, Ph. D. thesis, University of Glasgow, September 1971) by an X-ray study is deduced. Secondly, the vast field of nmr studies in conjugated systems is sketched relatively briefly before the few results relating to aromatic systems are detailed. Work on 9-nitroso, and 9-formyljulolidine rotation; :al barriers for the '9' group are then presented. Extensive electron delocalisation is observed from the nitrogen atom of the quinolizidine unit in these compounds to the '9' group and this is comparable to, or in excess of, that observed in para-substituted dimethylanilines. The rotational barriers for the dimethylamino function in three para-substituted N,N-dimethylanilines are then reported and a Hammett correlation between them is suggested. In this manner, an estimate of the rotational barrier for the dimethylamino function in N,N-dimethylaniline itself is presented. Upon this basis and upon results published for studies of aziridine inversion barriers, an estimate of the resonance energy in dimethylaniline is suggested at 10+/-1 kcal./mole. The necessary practical details of this work are then outlined. The importance of computing facilities in this work is made in the appendices and an original coding of a least squares method due to W. E. Wentworth (J. Chem. Ed. , 1965,42,96) is included. Published papers on this work are then appended

Holonomy and parallel transport for Abelian gerbes

In this paper we establish a one-to-one correspondence between $S^1$-gerbes with connections, on the one hand, and their holonomies, for simply connected manifolds, or their parallel transports, in the general case, on the other hand. This result is a higher-order analogue of the familiar equivalence between bundles with connections and their holonomies for connected manifolds. The holonomy of a gerbe with group $S^1$ on a simply connected manifold $M$ is a group morphism from the thin second homotopy group to $S^1$, satisfying a smoothness condition, where a homotopy between maps from $[0,1]^2$ to $M$ is thin when its derivative is of rank $\leq 2$. For the non-simply connected case, holonomy is replaced by a parallel transport functor between two monoidal Lie groupoids. The reconstruction of the gerbe and connection from its holonomy is carried out in detail for the simply connected case. Our approach to abelian gerbes with connections holds out prospects for generalizing to the non-abelian case via the theory of double Lie groupoids.Comment: Final version. Improved readibility (hopefully). LaTeX, 60 pages, 14 figures. To appear in Advances in Mathematic

The Binding of a Glycoprotein 120 V3 Loop Peptide to HIV-1 Neutralizing Antibodies STRUCTURAL IMPLICATIONS

Abstract The structural and antigenic properties of a peptide ("CRK") derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved "GPGR" residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II β-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK "GPGR" residues to adopt a β-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: "5023A" and "5025A," for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR β-turn structure

Binding characteristics of a panel of monoclonal antibodies against the ligand binding domain of the human LDLr

To obtain a panel of monoclonal antibodies (MAbs) to study the folding and conformation of the low density lipoprotein receptor (LDLr), we have generated hybridomas from LDLr-deficient mice that had been immunized with the extracellular domain of the human LDLr. The 12 MAbs were specific for the ligand binding domain of the LDLr, with individual MAbs recognizing epitopes in ligand binding repeats 1, 2, 3, 5, and 7. A subset of the MAbs failed to react with the LDLr when disulfide bonds were reduced, and one MAb, specific for an epitope that spans ligand binding repeats 1 and 2, recognized two conformational forms of the LDLr with different affinities. Antibodies specific for ligand binding repeats 3, 5, and 7 completely blocked the binding of LDL particles to the LDLr on cultured human fibroblasts, whereas MAbs with epitopes in ligand binding repeats 1 and 2 partially blocked the binding of LDL to the LDLr. These anti-LDLr MAbs will serve as useful probes for further analysis of LDLr conformation and LDLr-mediated lipoprotein binding

Intra-dance variation among waggle runs and the design of efficient protocols for honey bee dance decoding

Noise is universal in information transfer. In animal communication, this presents a challenge not only for intended signal receivers, but also to biologists studying the system. In honey bees, a forager communicates to nestmates the location of an important resource via the waggle dance. This vibrational signal is composed of repeating units (waggle runs) that are then averaged by nestmates to derive a single vector. Manual dance decoding is a powerful tool for studying bee foraging ecology, although the process is time-consuming: a forager may repeat the waggle run 1- >100 times within a dance. It is impractical to decode all of these to obtain the vector; however, intra-dance waggle runs vary, so it is important to decode enough to obtain a good average. Here we examine the variation among waggle runs made by foraging bees to devise a method of dance decoding. The first and last waggle runs within a dance are significantly more variable than the middle run. There was no trend in variation for the middle waggle runs. We recommend that any four consecutive waggle runs, not including the first and last runs, may be decoded, and we show that this methodology is suitable by demonstrating the goodness-of-fit between the decoded vectors from our subsamples with the vectors from the entire dances

Identification of a Cross-reactive Epitope Widely Present in Lipopolysaccharide from Enterobacteria and Recognized by the Cross-protective Monoclonal Antibody WN1 222-5

Septic shock due to infections with Gram-negative bacteria is a severe disease with a high mortality rate. We report the identification of the antigenic determinants of an epitope that is present in enterobacterial lipopolysaccharide (LPS) and recognized by a cross-reactive monoclonal antibody (mAb WN1 222-5) regarded as a potential means of treatment. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides obtained from Escherichia coli core types R1, R2, R3, and R4, Salmonella enterica, and the mutant strain E. coli J-5, we showed that mAb WN1 222-5 binds to the distal part of the inner core region and recognizes the structural element R1-alpha-d-Glcp-(1-->3)-[l-alpha-d-Hepp-(1-->7)]-l-alpha-d-Hepp 4P-(1-->3)-R2 (where R1 represents additional sugars of the outer core and R2 represents additional sugars of the inner core), which is common to LPS from all E. coli, Salmonella, and Shigella. WN1 222-5 binds poorly to molecules that lack the side chain heptose or lack phosphate at the branched heptose. Also molecules that are substituted with GlcpN at the side chain heptose are poorly bound. Thus, the side chain heptose and the 4-phosphate on the branched heptose are main determinants of the epitope. We have determined the binding kinetics and affinities (KD values) of the monovalent interaction of E. coli core oligosaccharides with WN1 222-5 by surface plasmon resonance and isothermal titration microcalorimetry. Affinity constants (KD values) determined by SPR were in the range of 3.6 x 10-5 to 3.2 x 10-8 m, with the highest affinity being observed for the core oligosaccharide from E. coli F576 (R2 core type) and the lowest KD values for those from E. coli J-5. Affinities of E. coli R1, R3, and R4 oligosaccharides were 5-10-fold lower, and values from the E. coli J-5 mutant were 29-fold lower than the R2 core oligosaccharide. Thus, the outer core sugars had a positive effect on binding

Evidence of Titan's Climate History from Evaporite Distribution

Water-ice-poor, 5-$\mu$m-bright material on Saturn's moon Titan has previously been geomorphologically identified as evaporitic. Here we present a global distribution of the occurrences of the 5-$\mu$m-bright spectral unit, identified with Cassini's Visual Infrared Mapping Spectrometer (VIMS) and examined with RADAR when possible. We explore the possibility that each of these occurrences are evaporite deposits. The 5-$\mu$m-bright material covers 1\% of Titan's surface and is not limited to the poles (the only regions with extensive, long-lived surface liquid). We find the greatest areal concentration to be in the equatorial basins Tui Regio and Hotei Regio. Our interpretations, based on the correlation between 5-$\mu$m-bright material and lakebeds, imply that there was enough liquid present at some time to create the observed 5-$\mu$m-bright material. We address the climate implications surrounding a lack of evaporitic material at the south polar basins: if the south pole basins were filled at some point in the past, then where is the evaporite

Disease associated polymorphisms within the conserved ECR1 enhancer differentially regulate the tissue specific activity of the cannabinoid‐1 receptor gene promoter; implications for cannabinoid pharmacogenetics

EH was funded by Medical Research Scotland (PhD-719-2013) and GW Pharmaceuticals. AMcE was funded by BBSRC project grant (BB/N017544/1). PB and DW are funded by the Scottish Government Rural and Environment Science and Analytical Services Division to the Rowett Institute. The authors declare no conflicts of interest.Peer reviewedPublisher PD

Analysis by Surface Plasmon Resonance of the Influence of Valence on the Ligand Binding Affinity and Kinetics of an Anti-carbohydrate Antibody

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer

Structure of an anti-blood group A Fv and improvement of its binding affinity without loss of specificity.

The specificity of antibody recognition of the ABO blood group trisaccharide antigens has been explored by crystal structure analysis and mutation methods. The crystal structure of the Fv corresponding to the anti-blood group A antibody AC1001 has been determined to 2.2-A resolution and reveals a binding pocket that is complementary to the blood group A-trisaccharide antigen. The effect of mutating specific residues lining this pocket on binding to the A and B blood group oligosaccharide antigens was investigated through a panel of single point mutations and through a phage library of mutations in complementarity determining region H3. Both approaches gave several mutants with improved affinity for antigen. Surface plasmon resonance indicated up to 8-fold enhancement in affinity for the A-pentasaccharide with no observable binding to the blood group B antigen. This is the first example of single point mutations in a carbohydrate-binding antibody resulting in significant increases in binding affinity without loss of specificity