Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The ' H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein ' H atoms, and the structure was calculated on the basis of 813 distance restraints from 'H-'H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 @-angles, 61 x' angles, and 2 x ' angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined a-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A f 0.04 A for backbone atoms and 0.93 A k 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur JBiochern 226:413-422; Shin DH et al, 1995, Structure3: 189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.

. A comparison of the structures of bLTP in the free and bound forms suggests that bLTP can accommodate long olefinic ligands by expansion of the hydrophobic binding site. This expansion is achieved by a bend of one helix, HA, and by conformational changes in both the C terminus and helix HC. This mode of binding is different from that seen in the structure of maize nsLTP in complex with palmitic acid, where binding of the ligand is not associated with structural changes.

Evaluation of the health related effects of beer intake is hampered by the lack of accurate tools for assessing intakes (biomarkers). Therefore, we identified plasma and urine metabolites associated with recent beer intake by untargeted metabolomics and established a characteristic metabolite pattern representing raw materials and beer production as a qualitative biomarker of beer intake. In a randomized, crossover, single-blinded meal study (MSt1), 18 participants were given, one at a time, four different test beverages: strong, regular, and nonalcoholic beers and a soft drink. Four participants were assigned to have two additional beers (MSt2). In addition to plasma and urine samples, test beverages, wort, and hops extract were analyzed by UPLC-QTOF. A unique metabolite pattern reflecting beer metabolome, including metabolites derived from beer raw material (i.e., N-methyl tyramine sulfate and the sum of iso-α-acids and tricyclohumols) and the production process (i.e., pyro-glutamyl proline and 2-ethyl malate), was selected to establish a compliance biomarker model for detection of beer intake based on MSt1. The model predicted the MSt2 samples collected before and up to 12 h after beer intake correctly (AUC = 1). A biomarker model including four metabolites representing both beer raw materials and production steps provided a specific and accurate tool for measurement of beer consumption.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has becn invcstigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phei Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.According to their substrate specificities, the endopeptidases can be divided into several groups. A number of endopeptidases, e.g. trypsin [3], have a very high specificity for particular amino acid residues at position PI, while others are less specific. In some of these enzymes with broadcr substrate preference, a binding subsite rcmote from the scissile bond is of primary importance, e.g. the S2 subsites of a-lytic protease [4] and papain [5], and the S4 subsites of elastase [6] and the subtilisins [7]. X-ray crystallographic studies of subtilisins BPN' and Carlsberg complexed with various inhibitor proteins [8 -121, and extensive kinetic characterisation of two subtilisins, subtilisin 309 from Bacillus lentils and subtilisin BPN' from Bacillus amyloliquefuciens [I 31, suggest that interaction between the substrate and the S4 binding pocket is at least as important for substrate preference as interaction in the S1 region.Subtilisin BPN' exhibits a marked preferencc for aromatic groups in P4 [13]; this has been claimed to be due to a tyrosine Enzyme. Subtilisin 309, a protease from Bacillus lentus (EC 3.4.21.14).Note. The binding-site notation is that of Schechter and Berger [I]. Accordingly, amino acid residues in the suhstrate are referred to as PI, Pz, . . . Pi and P;, P;, . . . Pi away from the scissile bond towards the N-and C-terminal amino acid residues of the substrate, respectively. Enzymc subsites are denoted S, , Sz, ... Si and S;, S;, ... SI in correspondcnce with the substrate. The numbering of amino acid residues is that of subtilisin BPN' from Bacillus miyloliquefaciens (21. residue, TyrlO4 located...

The amino acid side chains of Ile107, Leu126, and Leu135 participate in the formation of the important hydrophobic S4 binding pocket of the subtilisin Savinase. Ile107 and Leu126, located on each side of the pocket, point toward each other, and Leu135 is situated at the bottom of the pocket. These amino acid residues have been substituted for other hydrophobic amino acid residues by site-directed mutagenesis, and the resulting enzymes have been characterized with respect to their P4 substrate preferences. The Leu126-->Ala or Phe substitutions reduce kcat/KM for the hydrolysis of all substrates to around 5% without altering the substrate preference. It is concluded that Leu126 is an essential structural part of the pocket which cannot be replaced without seriously affecting catalysis, consistent with the fact that Leu126 is conserved among all subtilisins. In contrast, the Ile107-->Gly, Ala, Val, Leu, or Phe and Leu135-->Ala, Val, or Phe substitutions strongly influence the P4 substrate preference, and some of the mutants exhibit large specificity changes for particular substrates when compared to wild-type Savinase. The results can be rationalized on the basis of Ile107 and Leu135 being responsible for steric repulsion of branched aliphatic and aromatic P4 side chains, respectively. Leu135 exclusively interacts with aromatic P4 side chains, and its replacement with less bulky amino acid residues alleviates steric repulsion such that the activity toward this type of substrates is enhanced. Conversely, the introduction of a more bulky amino acid residue at position 135 produces more steric repulsion and reduces the activity toward substrates with aromatic P4 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)