Abbreviations: BLG, b-lactoglobulin. Note: the co-ordinates for variants A and B of b-lactoglobulin lattice Y have been deposited in the RCSB Protein Data Bank with accession nos 1qg5 and 1b8e, respectively. q FEBS 2001 Structures of bovine b-lactoglobulin A and B (Eur. J. Biochem. 268) 479 q FEBS 2001 Structures of bovine b-lactoglobulin A and B (Eur. J. Biochem. 268) 481 q FEBS 2001 Structures of bovine b-lactoglobulin A and B (Eur. J. Biochem. 268) 483

Effects of hydrostatic pressure on dimeric beta-lactoglobulin A (beta-Lg) were investigated. Application of pressures of up to 3.5 kbar induced a significant red shift ( approximately 11 nm) and a 60% increase in intrinsic fluorescence emission of beta-Lg. These changes were very similar to those induced by guanidine hydrochloride, which caused subunit dissociation and unfolding of beta-Lg. A large hysteresis in the recovery of fluorescence parameters was observed upon decompression of beta-Lg. Pressure-induced dissociation and unfolding were not fully reversible, because of the formation of a nonnative intersubunit disulfide bond that hampered correct refolding of the dimer. Comparison between pressure dissociation/unfolding at 3 degrees C and 23 degrees C revealed a marked destabilization of beta-Lg at low temperature. The stability of beta-Lg toward pressure was significantly enhanced by 1 M NaCl, but not by glycerol (up to 20% v/v). These observations suggest that salt stabilization was not related to a general cosolvent effect, but may reflect charge screening. Interestingly, pressure-induced dissociation/unfolding was completely independent of beta-Lg concentration, in apparent violation of the law of mass action. Possible causes for this anomalous behavior are discussed.

The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.

The amyloid precursor protein (APP) is the precursor of the beta-amyloid peptide (Abeta), which is centrally related to the genesis of Alzheimer's disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89% of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 A calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 A and 130 A, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30% of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed.

b-Lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of b-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471±476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of b-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DV unf ) of ± 49^8 mL´mol 21 and 275^3 mL´mol 21 , and unfolding free energy changes (DG unf ) of 8.5^1.3 kJ´mol 21 and 11.3^0.4 kJ´mol 21 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is < 40 A Ê 3 per monomer of b-lactoglobulin, in excellent agreement with the experimentally measured difference in DV unf for the two isoforms (DDV unf 26 mL´mol 21 , corresponding to < 43 A Ê 3 per monomer). Thus, the existence of a core cavity in b-lactoglobulin B may explain its enhanced pressure sensitivity relative to b-lactoglobulin A. b-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of b-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5^1.3 and 8.3^0.3 kJ´mol 21 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of b-lactoglobulin is identical at these pH values. Interestingly, DV unf was dependent on pH, and varied from 249^8 mL´mol 21 to 268^2 mL´mol 21 at pH 7 and 8, respectively. The large increase in DV unf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of b-lactoglobulin at alkaline pH.