Proteins conformation pressure

Solutions of macromolecules may be concentrated by means of polymer membranes of defined pore size. Applying a pressure or centrifugal force, small molecules pass the pores, whereas large molecules retain. The nominal cutoff of an ultrafiltration membrane (MWCO) helps you to select a membrane Molecules smaller than the MWCO will pass the membrane, whereas larger molecules are held back. This separation is not sharp and depends on protein conformation and solvent composition. Complete retention is achieved when using a membrane with a MWCO 1/3 to 1/5 of the molar mass of the macromolecule of interest. Figure 3.6 illustrates the separation of proteins by ultrafiltration. 

High hydrostatic pressure induces changes in protein conformation, solvation and enzyme activities via reversible and non-reversible effects on intra- and inter-molecular interactions (noncovalent bonds) [1]. To have access to these structural modifications, spectroscopic investigations are required which necessitate special spectroscopic adaptations. Two improvements are presented first for enzyme reactions and second for structural determination. 

Abstract. Walter Kauzmann stated in a review of protein thermodynamics that volume and enthalpy changes are equally fundamental properties of the unfolding process, and no model can be considered acceptable unless it accounts for the entire thermodynamic behaviour (Nature 325 763-764, 1987). While the thermodynamic basis for pressure effects has been known for some time, the molecular mechanisms have remained rather mysterious. We, and others in the rather small field of pressure effects on protein structure and stability, have attempted since that time to clarify the molecular and physical basis for the changes in volume that accompany protein conformational transitions, and hence to explain pressure effects on proteins. The combination of many years of work on a model system, staphylococcal nuclease and its large numbers of site-specific mutants, and the rather new pressure perturbation calorimetry approach has provided for the first time a fundamental qualitative understanding of AV of unfolding, the quantitative basis of which remains the goal of current work. 

A transition induced in the lipid bilayer will result in a conformation "pressure" on the protein so tiiat it can adapt to the changed bilayer structure. A consequence of this is the possibility of controlling lipid composition by an on/off switch of enzymes responsible for lipid modifications. For example the methyl transferase enzymes that convert phosphatidylethanolamines into phosphatidylcholines might be controlled in this way. (PE favours whereas PC favours the conformation and should thus be expected to switch on and switch off the enzyme activity). 

High hydrostatic pressure alters any process that proceeds with a volume change. This affects enzyme catalysis because both substrate binding and associated protein conformational changes proceed with changes in the hydration state of amino acid residues that alter the total volume of the water-protein-ligand system (Low and Somero, 1975). Therefore, animals adapted to the deep sea require changes to the amino acid composition of enzymes that create pressure-insensitive kinetic parameters and pressure-resistant structures. 

Pressure - Temperature Effects on Protein Conformational States... 

The role of water in the conformation, the activity and the stability of proteins has been investigated with many experimental and theoretical approaches. Because of its importance it has been coined as the 21 amino acid . There is now sufficient experimental evidence for the fact that dry proteins do not unfold by increased temperature or pressure [21]. Low levels of hydration give rise to a glassy state and the temperature of the glass transition depends on the amount of water as observed for synthetic polymers. Water can therefore be considered as a plasticizer of the protein conformation. Whereas hydrophobic interactions have dominated the interpretation of the data, hydrogen bond networks of water may also play a predominant role in water-mediated interactions [48,49]. 

More complex denaturation processes involving stable intermediates lead a to plurality of protein conformations, which can be probed by electrophoresis under pressure... 

So far there have been relatively few applications of electrophoresis at elevated hydrostatic pressures. However, this method has several advantages over other methods such as optical methods it is a simple and direct means of studying dissociation and denaturation processes, and of describing the thermodynamics of protein-ligand interactions. These qualitative and quantitative studies can be performed using small amounts of pure proteins or complex protein mixtures. In addition, this technique permits separation and subsequent isolation of the different protein conformational states or subunits. 

The above data indicate positive relationships between protein conformation, net hydrophobicity, surface pressure, surface yield stress, film elasticity, and foam stability. 

The structural analysis on KcsA was performed based on the mobility of each spin labeled side chain in the protein segments under investigation. It is worth recognizing in Pig. 4b that most of the CW RT spectra show multiple spectral components, characterized by different mobility (a few examples are highlighted by arrows). This is a very general property of the R1 side chain in proteins. The components reflect the anisotropy of the spin label reorientational motion, but their appearance could also have other causes. They could arise from a slow equilibrium between two different protein conformations or the presence of asymmetric sites in the protein. The molecular interpretation of different spectral components is cumbersome. Multifrequency EPR [17], temperature analysis of the CW spectra [27], pulse saturation recovery techniques [28], or high pressure EPR [29] can help unravel the possible origins of the spectral components. In the case of KcsA, the spin labels motional information was quantitatively extracted from the inverse central line width (A//q, mobility parameter) and was corroborated by the measure of the accessibility of the spin labeled side chains towards lipids (O2... 

K. Akasaka, Probing conformational fluctuation of proteins by pressure perturbation. Chem. Rev. Thematic Issue Protein Dynamics and Folding (Guest Editor Prof A.J.Wand), 2005. 

The partial molar volume is a thermodynamic quantity that plays an essential role in the analysis of pressure effects on chemical reactions, reaction rate as well as chemical equilibrium in solution. In the field of biophysics, the pressure-induced denaturation of protein molecules has continuously been investigated since an egg white gel was observed under the pressure of 7000 atmospheres [60]. The partial molar volume is a key quantity in analyzing such pressure effects on protein conformations When the pressure in increased, a change of the protein conformation is promoted in the direction that the partial molar volume reduces. A considerable amount of experimental work has been devoted to measuring the partial molar volume of a variety of solutes in many different solvents. However, analysis and interpretation of the experimental data are in many cases based on drastically simplified models of solution or on speculations without physical ground, even for the simplest solutes such as alkali-halide ions in aqueous solution. Matters become more serious when protein molecules featuring complicated conformations are considered. 

The 3D-RISM/HNC is free from the drawback mentioned above and gives more accurate values of the partial molar volume than the ID-RISM/HNC. However, the former is far more computationally demanding and hardly applicable to very large proteins. To make detailed analyses of the pressure effects on protein conformations possible, improvement of the ID-RISM/HNC is imperative. Based on our very recent studies [72], we believe that it is not difficult to achieve such improvement. 

Rll M. D. Collins, C. U. Kim and S. M. Gruner, High-Pressure Protein Crystallography and Nuclear Magnetic Resonance to Explore Protein Conformations , Annu. Rev. Biophys., 2011, 40, 81. 

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