Bovine β-lactoglobulin at 1.8 Å resolution — still an enigmatic lipocalin

Abstract Background: β -Lactoglobulin ( β -Lg) is the major whey protein in the milk of ruminants and many other mammals. Its function is not known, but it undergoes at least two pH-dependent conformational changes which may be important. Bovine β -Lg crystallizes in several different lattices, and medium-resolution structures of orthorhombic lattice Y and trigonal lattice Z have been published. Triclinic lattice X and lattice Z crystals grow at pH values either side of the pH at which one of the pH-induced conformational changes occurs. A full understanding of the structure is needed to help explain both the conformational changes and the different denaturation behaviour of the genetic variants. Results: We have redetermined the structure of β -Lg lattice Z at 3.0 A resolution by multiple isomorphous replacement and have partially refined it (R factor=24.8%). Using the dimer from this lattice Z structure as a search model, the triclinic crystal form grown at pH 6.5 (lattice X) has been solved by molecular replacement. Refinement of lattice X at 1.8 A resolution gave an R factor of 18.1%. The structure we have determined differs from previously published structures in several ways. Conclusions: Incorrect threading of the sequence in the published structures of β -Lg affects four of the nine β strands. The basic lipocalin fold of the polypeptide chain is unchanged, however. The relative orientation of the monomers in the β -Lg dimer differs in the two lattices. On raising the pH, there is a rotation of approximately 5°, which breaks a number of intersubunit hydrogen bonds. It is not yet clear, however, why the stability of the structure should depend so heavily upon the external loop around residue 64 or the β strand with the free thiol, each of which shows genetic variation.

[1]  P. Song,et al.  Spectroscopic characterization of β-lactoglobulin-retinol complex , 1980 .

[2]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[3]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[4]  K. Robillard,et al.  Aromatic hydrophobes and -lactoglobulin A. Thermodynamics of binding. , 1972, Biochemistry.

[5]  H. Swaisgood,et al.  Relative Structural Stabilities of .beta.-Lactoglobulins A and B as Determined by Proteolytic Susceptibility and Differential Scanning Calorimetry , 1994 .

[6]  D R Flower,et al.  The lipocalin protein family: structure and function. , 1996, The Biochemical journal.

[7]  E. M. Brown,et al.  Accessibility and mobility of lysine residues in beta-lactoglobulin. , 1988, Biochemistry.

[8]  L. Berliner,et al.  Fatty acids and retinoids bind independently and simultaneously to beta-lactoglobulin. , 1997, Biochemistry.

[9]  R. Huber,et al.  Molecular structure of the bilin binding protein (BBP) from Pieris brassicae after refinement at 2.0 A resolution. , 1987, Journal of molecular biology.

[10]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[11]  K. Brew,et al.  Homology of beta-lactoglobulin, serum retinol-binding protein, and protein HC. , 1985, Science.

[12]  T. Mcmeekin,et al.  The solubilities of β-lactoglobulins A, B and AB , 1964 .

[13]  C. Tanford,et al.  The Reversible Transformation of -Lactoglobulin at pH 7.5 , 1959 .

[14]  T. Attwood,et al.  Structure and sequence relationships in the lipocalins and related proteins , 1993, Protein science : a publication of the Protein Society.

[15]  G. I. Imafidon,et al.  Differential Scanning Calorimetric Study of Different Genetic Variants of β-Lactoglobulin , 1991 .

[16]  R. Aschaffenburg,et al.  Improved method for the preparation of crystalline β-lactoglobulin and α-lactalbumin from cow's milk , 1957 .

[17]  J. Mercier,et al.  Polymorphisme des lactoprotéines de bovinés népalais. I. – Mise en evidence, chez le yak, et caractérisation biochimique de deux nouveaux variants: β-lactoglobuline Dyak et caséine αs1E , 1976, Annales de génétique et de sélection animale.

[18]  C. Cambillau,et al.  Domain swapping creates a third putative combining site in bovine odorant binding protein dimer , 1996, Nature Structural Biology.

[19]  V S Lamzin,et al.  Automated refinement of protein models. , 1993, Acta crystallographica. Section D, Biological crystallography.

[20]  L. Sawyer,et al.  Expression and secretion of recombinant ovine beta-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis. , 1996, The Biochemical journal.

[21]  J. Findlay,et al.  The interaction of retinol-binding protein with its plasma-membrane receptor. , 1988, The Biochemical journal.

[22]  Z. Puhan,et al.  Technological properties of milk as influenced by genetic polymorphism of milk proteins — A review , 1992 .

[23]  R. Simmons,et al.  Crystal forms of -lactoglobulin , 1965 .

[24]  M. Totsuka,et al.  Tryptophan-19 of beta-lactoglobulin, the only residue completely conserved in the lipocalin superfamily, is not essential for binding retinol, but relevant to stabilizing bound retinol and maintaining its structure. , 1994, Biochimica et biophysica acta.

[25]  M. J. Gorbunoff.  Exposure of tyrosine residues in protein. Reaction of cyanuric fluoride with ribonuclease, alpha-lactalbumin, and beta-lactoglobulin. , 1967, Biochemistry.

[26]  P. A. Peterson,et al.  The three‐dimensional structure of retinol‐binding protein. , 1984, The EMBO journal.

[27]  Miguel Calvo Rebollar,et al.  Interaction of Fatty Acids with β-Lactoglobulin and Albumin from Ruminant Milk1 , 1989 .

[28]  T A Jones,et al.  Crystallographic refinement of human serum retinol binding protein at 2Å resolution , 1990, Proteins.

[29]  A. North,et al.  Three-dimensional arrangement of conserved amino acid residues in a superfamily of specific ligand-binding proteins. , 1989, International journal of biological macromolecules.

[30]  M. Barkley,et al.  Fluorescence quenching in indoles by excited-state proton transfer , 1992 .

[31]  R. Lyster,et al.  Whey protein denaturation in heated milk and cheese whey , 1979, Journal of Dairy Research.

[32]  A. North,et al.  Structure and function of bovine β-lactoglobulin , 1985 .

[33]  P. Main,et al.  The use of Sayre's equation with solvent flattening and histogram matching for phase extension and refinement of protein structures , 1990 .

[34]  C. Genot,et al.  beta-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy. , 1994, Biochimica et biophysica acta.

[35]  H. Baker,et al.  Structure of copper- and oxalate-substituted human lactoferrin at 2.0 A resolution. , 1994, Acta crystallographica. Section D, Biological crystallography.

[36]  G. H. McKenzie,et al.  Thermodenaturation of bovine -lactoglobulin. Kinetics and the introduction of -structure. , 1971, Biochimica et biophysica acta.

[37]  R. Aschaffenburg,et al.  Occurrence of Different Beta-Lactoglobulins in Cow's Milk , 1955, Nature.

[38]  D. Eisenberg,et al.  Assessment of protein models with three-dimensional profiles , 1992, Nature.

[39]  L. Sawyer.  One fold among many , 1987, Nature.

[40]  S. N. Timasheff,et al.  The state of amino acid residues in β-lactoglobulin , 1969 .

[41]  W. Sawyer.  Heat Denaturation of Bovine β-Lactoglobulins and Relevance of Disulfide Aggregation , 1968 .

[42]  D. W. Green,et al.  Sulphydryl groups and the N⇄R conformational change in β-lactoglobulin , 1966 .

[43]  G. Kleywegt,et al.  Halloween ... Masks and Bones , 1994 .

[44]  Wolfgang Kabsch,et al.  Automatic indexing of rotation diffraction patterns , 1988 .

[45]  Pace Cn,et al.  A comparison of the denaturation of bovine -lactoglobulins A and B and goat -lactoglobulin. , 1971 .

[46]  M. Newcomer.  Structure of the epididymal retinoic acid binding protein at 2.1 A resolution. , 1993, Structure.

[47]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[48]  K. Brew,et al.  Homology and structure‐function correlations between α1 ‐acid glycoprotein and serum retinol‐binding protein and its relatives , 1987, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[49]  R. Jenness,et al.  Heat Denaturation of β-Lactoglobulins A and B , 1962 .

[50]  D. Green.  Structure of bovine ?-lactoglobulin at 6p resolution , 1979 .

[51]  E. Baker,et al.  Hydrogen bonding in globular proteins. , 1984, Progress in biophysics and molecular biology.

[52]  A T Brünger,et al.  Slow-cooling protocols for crystallographic refinement by simulated annealing. , 1990, Acta crystallographica. Section A, Foundations of crystallography.

[53]  O. Campanella,et al.  Thermal gelation and denaturation of bovine β-lactoglobulins A and B , 1994, Journal of Dairy Research.

[54]  M. Behe,et al.  Binding of p-nitrophenyl phosphate and other aromatic compounds by beta-lactoglobulin. , 1987, Journal of dairy science.

[55]  McKenzie Ha,et al.  Location of sulfhydryl and disulfide groups in bovine -lactoglobulins and effects of urea. , 1972 .

[56]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[57]  A. North,et al.  Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography , 1992, Nature.

[58]  M. Bolognesi,et al.  Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 A resolution. , 1987, Journal of molecular biology.

[59]  T A Jones,et al.  The three‐dimensional structure of P2 myelin protein. , 1988, The EMBO journal.

[60]  Barry C. Finzel,et al.  The use of an imaging proportional counter in macromolecular crystallography , 1987 .

[61]  P. Kraulis,et al.  The structure of β-lactoglobulin and its similarity to plasma retinol-binding protein , 1986, Nature.