Stanislava Uhrinova

Complete assignment of 1H, 13C and 15N chemical shifts for bovine beta-lactoglobulin: secondary structure and topology of the native state is retained in a partially unfolded form.

Although β-lactoglobulin (β-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine β-LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of β-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a13 C,15N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric β-LG A. It includes eight antiparallel β-strands arranged in a barrel, flanked by an α-helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth β-strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of β-LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.

Solution structure of a functionally active fragment of decay-accelerating factor

The second and third modules of human decay accelerating factor (DAF) are necessary and sufficient to accelerate decay of the classical pathway (CP) convertase of complement. No structure of a mammalian protein with decay-accelerating activity has been available to date. We therefore determined the solution structure of DAF modules 2 and 3 (DAF∼2,3). Structure-guided analysis of 24 mutants identified likely contact points between DAF and the CP convertase. Three (R96, R69, and a residue in the vicinity of L171) lie on DAF∼2,3s concave face. A fourth, consisting of K127 and nearby R100, is on the opposite face. Regions of module 3 remote from the semiflexible 2–3 interface seem not to be involved in binding to the CP convertase. DAF thus seems to occupy a groove on the CP convertase such that both faces of DAF close to the 2–3 junction (including a positively charged region that encircles the protein at this point) interact simultaneously. Alternative pathway convertase interactions with DAF require additional regions of CCP 3 lying away from the 2–3 interface, consistent with the established additional requirement of module 4 for alternative pathway regulation.

Milk protein structure - what can it tell the dairy industry?

This paper describes the possible usefulness of knowledge of the three-dimensional structure of milk proteins to dairy scientists. After a brief introduction of the available methodology and the structures of milk proteins that are already available, the structure of bovine β-lactoglobulin is used to illustrate the possible applications of the structure to understanding the problems to which the protein contributes during milk processing.

Backbone dynamics of complement control protein (CCP) modules reveals mobility in binding surfaces.

The regulators of complement activation (RCA) are critical to health and disease because their role is to ensure that a complement‐mediated immune response to infection is proportionate and targeted. Each protein contains an uninterrupted array of from four to 30 examples of the very widely occurring complement control protein (CCP, or sushi) module. The CCP modules mediate specific protein–protein and protein–carbohydrate interactions that are key to the biological function of the RCA and, paradoxically, provide binding sites for numerous pathogens. Although structural and mutagenesis studies of CCP modules have addressed some aspects of molecular recognition, there have been no studies of the role of molecular dynamics in the interaction of CCP modules with their binding partners. NMR has now been used in the first full characterization of the backbone dynamics of CCP modules. The dynamics of two individual modules—the 16th of the 30 modules of complement receptor type 1 (CD35), and the N‐terminal module of membrane cofactor protein (CD46)—as well as their solution structures, are compared. Although both examples share broadly similar three‐dimensional structures, many structurally equivalent residues exhibit different amplitudes and timescales of local backbone motion. In each case, however, regions of the module‐surface implicated by mutagenesis as sites of interactions with other proteins include several mobile residues. This observation suggests further experiments to explore binding mechanisms and identify new binding sites.

Letter to the Editor: Resonance assignments of the central complement control protein module pair of human decay accelerating factor

Letter to the Editor: Resonance assignments of the central complement control protein module pair of human decay accelerating factor Stanislava Uhrinova1, Feng Lin2, Dusan Uhrin1, M. Edward Medof2 & Paul N. Barlow1,∗ 1The Edinburgh Protein Interaction Centre, University of Edinburgh, Edinburgh EH9 3JJ, Scotland, U.K.; 2Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, U.S.A.