Peter Gettins

Structure of an antibody combining site by magnetic resonance.

The structure of the combining site of the DNP binding IgA mouse myeloma protein MOPC 315 has been determined by a combination of high resolution nuclear magnetic resonance, electron spin resonance, model building and chemical modifications. This approach yields the general dimensions of the site, its polarity and asymmetry features, the assignment of the DNP-contact residues and their three-dimensional coordinates.

A physico-chemical study of heparin: Evidence for a calcium-induced co-operative conformational transition

Abstract The conformations of heparin in aqueous solution in the presence of sodium, potassium, magnesium and calcium cations were studied using circular dichroism, optical rotation, nuclear magnetic resonance and equilibrium dialysis. Potassium and magnesium cations, when added to sodium heparinate solutions, cause small chiroptical changes. Binding of calcium ions gives rise to large changes in both optical rotation and circular dichroism. This is indicative of a major change in chain conformation, which is also manifest in 13C and 1H n.m.r. § Equilibrium dialysis suggests one mole of calcium bound per mole of tetrasaccharide, which n.m.r. indicates to be appropriately sulphated iduronateglucosamine-iduronate-glucosamine. The calcium is chelated by two iduronate carboxyl groups. Proton-proton coupling constants, determined by convolution difference spectroscopy and Carr-Purcell sequences, indicate that, over the temperature range 285 to 353 K, the iduronate ring is best described as 1C4( l ) and the glucosamine residue as 4C1( d ) for both sodium and calcium forms. The conformational change induced by calcium is ascribed to rotation around the glycosidic linkages. The binding process is co-operative and the binding constant of 103 to 104 m −1 is biologically significant. The findings are consistent with intramolecular binding. Hence, this study represents the first report of a polysaccharide undergoing a cation-induced intramolecular disorder-order process. The authors postulate that a function of the post-polymerization epimerization of d -glucuronate to l -iduronate is the attainment of the precise geometry required for co-operative calcium binding with consequent modulation of the flexibility of the tetrasaccharide units.

Strategies for spectral assignment in the 1H NMR spectra of a 25 000 Mr murine antibody fragment: (i) in vivo deuteration and (ii) use of a denaturant

The Fv fragment of M3 15 is a murine antibody fragment of M, 25 000, which possesses high affinity for nitroaromatic haptens. It has been studied extensively by high resolution ‘I-l NMR in an attempt to understand the interactions and perturbations on antigen binding [l-3]. Large upfield changes in chemical shift of the hapten resonances were used to deduce a structure for the combining site. However, because no assignments, other than histidines, had been made, data on the changes in chemical shift of antibody resonances could only be used in a very qualitative way to state that different DNP and TNP haptens bind very similarly to the protein. In a protein of this size spectral assignment is extremely difficult because no spin-spin coupling is visible, This results from the large linewidths and the overlap of many resonances. The most satisfactory spectral simplification is to replace selected amino acids by deuterated analogues, since, unlike, e.g., lvF substitution, the resulting chemically modified system can be described realistically as unperturbed. Extensive rather than specific deuteration to simplify the ‘H NMR spectrum has been widely applied [4-8]. These studies have, however, all been confined to bacterial proteins, where a reasonably high yield of the desired protein can be expected. We report here the first instance of specific deuteration in a mammalian system. Tryptophan deuterated at all

Investigation of hapten—antibody interactions in McPC603 by 1H and 31P NMR spectroscopy

With the determination of the three dimensional structures of two Fab fragments [1,2] by X-ray crystallography and of the Dnp binding site of an Fv fragment by NMR [3], our understanding of the molecular basis of antibody-antigen recognition has been considerably advanced. It is now realised that there is a high degree of complementarity between the different functional moieties of the hapten and the three dimensional distribution of amino acids in the combining site, all of which come from the small percentage of residues designated as hypervariable [4]. Although the structure of the combining site of the phosphoryl choline-binding antibody McPC603 has been largely determined by X-ray crystallography [5], questions which remain unanswered are the importance of interactions between the phosphate moiety and the side chains of Arg 52 a and Tyr 33 H and whether the hapten is bound as the monoor dianion. This paper is concerned with the use of NMR spectroscopy to answer this by unambiguously identifying the phosphorus species involved in binding and to do this in solution, thereby providing dynamic as well as structural information. In addition, the crystal structure is used as a means of interpreting high resolution 1H NMR difference spectra obtained on addition ofhapten. This is important for the extension of NMR to the solving of unknown protein structures, by obtaining at least empirical agreement between the predicted and observed effects of aromatic and other perturbing residues. 2. Materials and methods

Nitrogen-15 nuclear magnetic resonance spectroscopy as a probe of hapten--antibody interactions: 15N-enriched trinitrophenyl haptens binding to M315.

One of the major contributions to the affinity of dinitrophenyland trimtrophenyl-binding antibodies is thought to arise from interactions between the protein and the nitro groups [ 11. It was concluded from a thermodynamic study [2] on the myeloma protein M3 15 that hydrogen-bond~g is involved in this interaction. A more direct ~vestigation by resonance Raman spectroscopy on the same protein suggested, however, that the nitro group of dinitrophenyl haptens with different sidechains interact differently in the dinitrophenyl binding site [3]. “N NMR provides an alternative approach for the direct study of interactions between antibodies and the nitro groups of such haptens. We wish to report the first application of this method to the study of the binding of specifically enriched trinitrophenyl haptens(without large side chains) to the Fv fragment of the dinitroand tr~itro-phenyl~~d~g myeloma M3 15.

Research lettersThiol ester role in correct folding and conformation of human α2-macroglobulin: Properties of recombinant C949S variant

To determine the role of the thiol ester in the folding of human alpha 2-macroglobulin (alpha 2M) in the active conformation, we have characterized a recombinant variant of alpha 2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S alpha 2M behaves like methylamine-treated plasma alpha 2M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric alpha 2M does not require a pre-formed thiol ester. Active alpha 2M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S alpha 2M followed by thiol ester formation and a conformational change that gives the native active state.To determine the role of the thiol ester in the folding of human α2‐macroglobulin (α2M) in the active conformation, we have characterized a recombinant variant of α2M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester‐forming cysteine. C949S α2M behaves like methylamine‐treated plasma α2M, with correctly formed inter‐subunit disulfide bridges, non‐covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric α2M does not require a pre‐formed thiol ester. Active α2M may form in vivo by a two‐step process involving initial folding to give a structure resembling that of C949S α2M followed by thiol ester formation and a conformational change that gives the native active state.