Compressibility as a means to detect and characterize globular protein states.

We report compressibility data on single-domain, globular proteins which suggest a general relationship between protein conformational transitions and delta kzeroS, the change in the partial specific adiabatic compressibility which accompanies the transition. Specifically, we find transitions between native and compact intermediate states to be accompanied by small increases in kzeroS of +(1-4) x 10(-6) cm3.g-1.bar-1 (1 bar = 100 kPa). By contrast, transitions between native and partially unfolded states are accompanied by small decreases in kzeroS of -(3-7) x 10(-6) cm3.g-1.bar-1, while native-to-fully unfolded transitions result in large decreases in kzeroS of -(18-20) x 10(-6) cm3.g-1.bar-1. Thus, for the single-domain, globular proteins studied here, changes in kzeroS correlate with the type of transition being monitored, independent of the specific protein. Consequently, kzeroS measurements may provide a convenient approach for detecting the existence of and for defining the nature of protein transitions, while also characterizing the hydration properties of individual protein states.

The structure of a PII signaling protein from a halophilic archaeon reveals novel traits and high-salt adaptations

To obtain insights into archaeal nitrogen signaling and haloadaptation of the nitrogen/carbon/energy-signaling protein PII, we determined crystal structures of recombinantly produced GlnK2 from the extreme halophilic archaeon Haloferax mediterranei, complexed with AMP or with the PII effectors ADP or ATP, at respective resolutions of 1.49 Å, 1.45 Å, and 2.60 Å. A unique trait of these structures was a three-tongued crown protruding from the trimer body convex side, formed by an 11-residue, N-terminal, highly acidic extension that is absent from structurally studied PII proteins. This extension substantially contributed to the very low pI value, which is a haloadaptive trait of H. mediterranei GlnK2, and participated in hexamer-forming contacts in one crystal. Similar acidic N-extensions are shown here to be common among PII proteins from halophilic organisms. Additional haloadaptive traits prominently represented in H. mediterranei GlnK2 are a very high ratio of small residues to large hydrophobic aliphatic residues, and the highest ratio of polar to nonpolar exposed surface for any structurally characterized PII protein. The presence of a dense hydration layer in the region between the three T-loops might also be a haloadaptation. Other unique findings revealed by the GlnK2 structure that might have functional relevance are: the adoption by its T-loop of a three-turn α-helical conformation, perhaps related to the ability of GlnK2 to directly interact with glutamine synthetase; and the firm binding of AMP, confirmed by biochemical binding studies with ATP, ADP, and AMP, raising the possibility that AMP could be an important PII effector, at least in archaea.

Nutrient partitioning during treatment of tuberculosis: gain in body fat mass but not in protein mass

Background: Tuberculosis is an important cause of wasting. The functional consequences of wasting and recovery may depend on the distribution of lost and gained nutrient stores between protein and fat masses. Objective: The goal was to study nutrient partitioning, ie, the proportion of weight change attributable to changes in fat mass (FM) versus protein mass (PM), during anti mycobacterial treatment. Design: Body-composition measures were made of 21 men and 9 women with pulmonary tuberculosis at baseline and after 1 and 6 mo of treatment. All subjects underwent dual-energy X-ray absorptiometry and deuterium bromide dilution tests, and a four-compartment model of FM, total body water (TBW), bone minerals (BM), and PM was derived. The ratio of PM to FM at any time was expressed as the energy content (p-ratio). Changes in the p-ratio were related to disease severity as measured by radiologic criteria. Results: Patients gained 10% in body weight (P < 0.001) from baseline to month 6. This was mainly due to a 44% gain in FM (P < 0.001); PM, BM, and TBW did not change significantly. Results were similar in men and women. The p-ratio decreased from baseline to month 1 and then fell further by month 6. Radiologic disease severity was not correlated with changes in the p-ratio. Conclusions: Microbiological cure of tuberculosis does not restore PM within 6 mo, despite a strong anabolic response. Change in the p-ratio is a suitable parameter for use in studying the effect of disease on body composition because it allows transformation of such effects into a normal distribution across a wide range of baseline proportion between fat and protein mass.

Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method

The ann of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous Solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P < 0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles < liposomes and lipidic/layered structures < ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P < 0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems. (C) 2004 Elsevier B.V. All rights reserved.

Quil A-lipid powder formulations releasing ISCOMs and related colloidal stuctures upon hydration

The aim of the present study was to prepare solid Quil A-cholesterol-phospholid formulations (as powder mixtures or compressed to pellets) by physical mixing or by freeze-drying of aqueous dispersions of these components in ratios that allow spontaneous formation of ISCOMs and other colloidal stuctures upon hydration. The effect of addition of excess cholesterol to the lipid mixtures on the release of a model antigen (PE-FITC-OVA) from the pellets was also investigated. Physical properties were evaluated by X-ray powder diffractometry (XPRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and polarized light microscopy (PLM). Characterization of aqueous colloidal dispersions was performed by negative staining transmission electron microscopy (TEM). Physically mixed powders (with or without PE-FITC-OVA) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures such as worm-like micelles, ring-like micelles, lipidic/layered structures and lamellae (hexagonal array of ring-like micelles) upon hydration as expected from the pseudo-temary diagram for aqueous mixtures of Quil A, cholesterol and phospholipid. In contrast, spontaneous formation of the expected colloids was demonstrated for the freeze-dried lipid mixtures. Pellets prepared by compression of freeze-dried powders released PE-FITC-OVA slower than those prepared from physically mixed powders. TEM investigations revealed that the antigen was released in the form of colloidal particles (ISCOMs) from pellets prepared by compression of freeze-dried powders. The addition of excess cholesterol slowed down the release of antigen. The findings obtained in this study are important for the formulation of solid Quil A-containing lipid articles as controlled particulate adjuvant containing antigen delivery systems. (c) 2004 Elsevier B.V. All rights reserved.

Protein interactions studied by SAXS:effect of ionic strength and protein concentration for BSA in aqueous solutions

We have studied a series of samples of bovine serum albumin (BSA) solutions with protein concentration, c, ranging from 2 to 500 mg/mL and ionic strength, I, from 0 to 2 M by small-angle X-ray scattering (SAXS). The scattering intensity distribution was compared to simulations using an oblate ellipsoid form factor with radii of 17 x 42 x 42 A, combined with either a screened Coulomb, repulsive structure factor, S-SC(q), or an attractive square-well structure factor, S-SW(q). At pH = 7, BSA is negatively charged. At low ionic strength, I <0.3 M, the total interaction exhibits a decrease of the repulsive interaction when compared to the salt-free solution, as the net surface charge is screened, and the data can be fitted by assuming an ellipsoid form factor and screened Coulomb interaction. At moderate ionic strength (0.3-0.5 M), the interaction is rather weak, and a hard-sphere structure factor has been used to simulate the data with a higher volume fraction. Upon further increase of the ionic strength (I >= 1.0 M), the overall interaction potential was dominated by an additional attractive potential, and the data could be successfully fitted by an ellipsoid form factor and a square-well potential model. The fit parameters, well depth and well width, indicate that the attractive potential caused by a high salt concentration is weak and long-ranged. Although the long-range, attractive potential dominated the protein interaction, no gelation or precipitation was observed in any of the samples. This is explained by the increase of a short-range, repulsive interaction between protein molecules by forming a hydration layer with increasing salt concentration. The competition between long-range, attractive and short-range, repulsive interactions accounted for the stability of concentrated BSA solution at high ionic strength.

Studies of Protein-Protein and Protein-Water Interactions by Small Angle X-Ray Scattering, Terahertz Spectroscopy, ASMOS, And Computer Simulation

The protein folding problem has been one of the most challenging subjects in biological physics due to its complexity. Energy landscape theory based on statistical mechanics provides a thermodynamic interpretation of the protein folding process. We have been working to answer fundamental questions about protein-protein and protein-water interactions, which are very important for describing the energy landscape surface of proteins correctly. At first, we present a new method for computing protein-protein interaction potentials of solvated proteins directly from SAXS data. An ensemble of proteins was modeled by Metropolis Monte Carlo and Molecular Dynamics simulations, and the global X-ray scattering of the whole model ensemble was computed at each snapshot of the simulation. The interaction potential model was optimized and iterated by a Levenberg-Marquardt algorithm. Secondly, we report that terahertz spectroscopy directly probes hydration dynamics around proteins and determines the size of the dynamical hydration shell. We also present the sequence and pH-dependence of the hydration shell and the effect of the hydrophobicity. On the other hand, kinetic terahertz absorption (KITA) spectroscopy is introduced to study the refolding kinetics of ubiquitin and its mutants. KITA results are compared to small angle X-ray scattering, tryptophan fluorescence, and circular dichroism results. We propose that KITA monitors the rearrangement of hydrogen bonding during secondary structure formation. Finally, we present development of the automated single molecule operating system (ASMOS) for a high throughput single molecule detector, which levitates a single protein molecule in a 10 µm diameter droplet by the laser guidance. I also have performed supporting calculations and simulations with my own program codes.