Investigation of high pressure effect on the structure and adsorption of ß-lactoglobulin.

ß-Lactoglobulin, being one of the principal whey protein, is of huge importance to the food industry. Temperature/pressure effects on this small protein has been extensively studied by industry. To characterize biochemical properties of ß-lactoglobulin after or during pressurization, a wide range of methods have been used thus far. In this study, for the first time, the pressure-induced conformation of ß-lactoglobulin in the crystal state was determined, at pressure 430 MPa. Changes observed in the high pressure structure correlate with the physico-chemical properties of pressure-treated ß-lactoglobulin obtained from dynamic light scattering, electrophoretic mobility and quartz crystal microbalance with dissipation monitoring measurements. A comparison between the ß-lactoglobulin structures determined at both high and ambient pressure contrasts the stable nature of the protein core and adjacent loop fragments. At high pressure the ß-lactoglobulin structure presents early signs of dimer dissociation, charge and conformational changes characteristic for initial unfolded intermediate as well as a significant modification of the binding pocket volume. Those observations are supported by changes in zeta potential values and results in increase affinity of the ß-lactoglobulin adsorption onto gold surface. Observed pressure-induced structural modifications were previously suggested as an important factor contributing to ß-lactoglobulin denaturation process. Presented studies provide detailed analysis of pressure-associated structural changes influencing ß-lactoglobulin conformation and consequently its adsorption.

Impact of ligand and protein desolvation on ligand binding to the S1 pocket of thrombin.

In the present study, we investigate the impact of a tightly bound water molecule on ligand binding in the S1 pocket of thrombin. The S1 pocket contains a deeply buried deprotonated aspartate residue (Asp189) that is, due to its charged state, well hydrated in the uncomplexed state. We systematically studied the importance of this water molecule by evaluating a series of ligands that contains pyridine-type P1 side chains that could potentially alter the binding properties of this water molecule. All of the pyridine derivatives retain the original hydration state albeit sometimes with a slight perturbance. In order to prevent a direct H-bond formation with Asp189, and to create a permanent positive charge on the P1 side chain that is positioned adjacent to the Asp189 carboxylate anion, we methylated the pyridine nitrogen. This methylation resulted in displacement of water but was accompanied by a loss in binding affinity. Quantum chemical calculations of the ligand solvation free energy showed that the positively charged methylpyridinium derivatives suffer a large penalty of desolvation upon binding. Consequently, they have a substantially less favorable enthalpy of binding. In addition to the ligand desolvation penalty, the hydration shell around Asp189 has to be overcome, which is achieved in nearly all pyridinium derivatives. Only for the ortho derivative is a partial population of a water next to Asp189 found. Possibly, the gain of electrostatic interactions between the charged P1 side chain and Asp189 helps to compensate for the desolvation penalty. In all uncharged pyridine derivatives, the solvation shell remains next to Asp189, partly mediating interactions between ligand and protein. In the case of the para-pyridine derivative, a strongly disordered cluster of water sites is observed between ligand and Asp189.

Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S1'-pocket of thermolysin.

BACKGROUND: Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S1'-pocket. METHODS: A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry. RESULTS AND CONCLUSIONS: Binding affinity increases with larger ligand hydrophobic P1'-moieties accommodating the S1'-pocket. Surprisingly, larger P1'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S1'-pocket or the formation of apolar interactions between protein and inhibitor. GENERAL SIGNIFICANCE: Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding.

The tobacco plasma membrane aquaporin NtAQP1.

This paper gives a summary of a project to characterize a tobacco aquaporin. The cDNA and gene, including the 5' upstream region, for the tobacco aquaporin NtAQP1 has been isolated and the encoded protein characterized. The significance of promoter regions for an abscisic acid- and gibberellic acid-induced gene expression could be restricted to a region between -1450 and -1112 upstream of the transcription start point by transient transformation of a bicistronic vector into tobacco protoplasts. NtAQP1 expression in tobacco plants was found to be elevated in flowers, stems and roots. In roots, the protein was detected close to xylem vessels in pitch-like structures. Studies with a NtAQP1-GFP fusion indicated a plasma membrane location. For a functional analysis, the cDNA was expressed in Xenopus oocytes. NtAQP1 was found to be a heavy metal-insensitive aquaporin with additional permeability for glycerol. Mutation of a threonine at position 233 to a cysteine transformed NtAQP1 into a heavy metal-sensitive aquaporin.

Arbuscular mycorrhiza development regulates the mRNA abundance of Mtaqp1 encoding a mercury-insensitive aquaporin of Medicago truncatula.

The genome of the model legume Medicago truncatula Gaertn. was screened for the presence of genes encoding tonoplast intrinsic proteins, and a gene family was identified. The cDNA fragments of two members of the multigene family were cloned from roots inoculated with an arbuscular mycorrhizal fungus. Transcript accumulation in roots could be detected for both cDNA fragments, but only one gene was induced in the symbiosis when compared to non-mycorrhizal control roots. A full-length cDNA clone was obtained from the arbuscular-mycorrhiza-regulated gene, and injection of in-vitro-transcribed RNA into Xenopus oocytes revealed that the encoded protein MtAQP1 specifically facilitates water transport. The possible role of MtAQP1 in buffering osmotic fluctations in the highly compartmented vacuole of arbuscule cells is discussed.

The Nicotiana tabacum plasma membrane aquaporin NtAQP1 is mercury-insensitive and permeable for glycerol.

A new aquaporin from Nicotiana tabacum (cv. Samsun) was characterized. It shares sequence homology to the Arabidopsis thaliana PIP1 protein family. By two-phase partitioning and immunoblot analysis, plasma membrane localization could be demonstrated. The corresponding mRNA is highly abundant in roots and flowers, while it is rarely expressed in leaves and stems. Functional expression in Xenopus oocytes revealed that NtAQP1 can mediate glycerol transport in addition to water flow. However, NtAQP1 is impermeable for Na+, K+ and Cl- ions. The water permeability and selectivity could not be modulated by addition of mercurials or the activity of cAMP-dependent protein kinases.

Cognitive Schemas in Social Perception.

This paper explores the cognitive schemas involved in ingroup and outgroup perceptions of three U. S. religious denominations (Baptists, Lutherans, and Catholics) based on a factor analytically derived rating scale. Two principal techniques are used for assessing these perceptions, that is, Similarity Structure Analysis, SSA, and Procrustean Individual Differences Scaling, PINDIS. The first is used to determine what the within-group and between-group perceptual structures are, while the latter is used to determine communalities among the various structures (stereotypy). Results revealed a high communality between denominational self-perceptions as well as some stereotypic misperceptions of Baptists and Catholics, but not of Lutherans.