The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

We report here, the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A. Concanavalin A from Canavalia ensiformis is a protein containing 237 amino acid residues with each monomer possessing one sugar binding site as well as sites for transition-metal ions, Mn2+ and Ca2+. The lectin binds specifically to α-anomers of monosaccharides, d-glucopyranoside and d-mannopyranoside, and recognizes the trimannosidic core of N-linked glycoproteins, 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside with high specificity, which constitutes the minimum carbohydrate epitope that completely fills the sugar binding site. Sensitive isothermal titration microcalorimetry coupled with osmotic stress strategy on concanavalin A was used to dissect out the differential involvement of water molecules in the recognition of the branched trimannoside (3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside), the individual dimannosidic arms (3-O-(α-d-mannopyranosyl)-α-d-mannopyranoside and 6-O-(α-d-mannopyranosyl)-α-d-mannopyranoside) as well as the monomer unit, d-mannopyranoside. The specific binding of concanavalin A to different sugars, is accompanied by differential uptake of water molecules during the binding process. These results not only complement the X-ray crystallographic studies of legume lectin−sugar complexes displaying structurally conserved water molecules mediating the specific ligation of the sugars with the corresponding sites in the binding pocket but also provide a rationale for the observed compensatory behavior of enthalpies with entropies in lectin−sugar interactions.

The innate immune system constitutes the first line of defense against microorganisms in both vertebrates and invertebrates. Although much progress has been made toward identifying key receptors and understanding their role in host defense, far less is known about how these receptors recognize microbial ligands. Such studies have been severely hampered by the need to purify ligands from microbial sources and a reliance on biological assays, rather than direct binding, to monitor recognition. We used synthetic peptidoglycan (PGN) derivatives, combined with microcalorimetry, to define the binding specificities of human and insect peptidogycan recognition proteins (PGRPs). We demonstrate that these innate immune receptors use dual strategies to distinguish between PGNs from different bacteria: one based on the composition of the PGN peptide stem and another that senses the peptide bridge crosslinking the stems. To pinpoint the site of PGRPs that mediates discrimination, we engineered structure-based variants having altered PGN-binding properties. The plasticity of the PGRPbinding site revealed by these mutants suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges.affinity ͉ bacteria ͉ innate immunity ͉ calorimetry ͉ synthesis T he innate immune system recognizes invading microbes by means of conserved pattern recognition receptors that bind unique products of microbial metabolism not produced by the host (pathogen-associated molecular patterns) (1, 2). Examples of microbial ligands recognized by pattern recognition receptors such as Toll-like receptors, peptidoglycan recognition proteins (PGRPs), and NOD proteins include lipopolysaccharide of Gram-negative bacteria, lipoteichoic acid of Gram-positive bacteria, nonmethylated CpG sequences, flagellin, and peptidoglycan (PGN) of Gram-negative and -positive bacteria. Cellular activation by pattern recognition receptors results in acute inflammatory responses involving cytokine and chemokine production, direct local attack against the invading pathogen, and induction of the adaptive component of the immune system. In humans, overactivation of inflammatory responses can lead to septic shock, which accounts for 100,000 deaths annually in the United States alone. By sitting at the intersection of the pathways of microbial recognition, inflammation, and cell death, the innate immune system offers emerging opportunities for the development of therapeutics to modulate immune responses (3).PGRPs, a newly discovered class of pattern recognition receptors, are highly conserved from insects to mammals (4-7). By detecting PGN from both Gram-negative and -positive bacteria, PGRPs are important contributors to host defense against microbial infections (2, 4). PGNs are polymers of alternating Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) in ␤(134) linkage, crosslinked by short peptide stems composed of alternating L-and D-amino acids (8, 9) (Fig. 1A). Whereas the carbohydrate backbone is conserved a...