Protein partition between the different phases comprising poly(ethylene glycol)-salt aqueous two-phase systems, hydrophobic interaction chromatography and precipitation: a generic description in terms of salting-out effects

The solution behaviour of selected proteins has been studied under conditions promoting precipitation, binding to mildly hydrophobic adsorbents or partition. Solvophobic theory may be used to describe these forms of protein partition. The tendency of a protein to partition therein is dependent upon surface properties of the protein solute mediated by the concentration and nature of added salts. As applied to partitioning in poly(ethylene glycol) (PEG)-salt systems this implies that linear (Brönsted) relationships apply only to proteins partitioned close to the critical point. At longer tie-line lengths protein partitioning is increasingly influenced by salting-out forces. This is confirmed by the observed behaviour of the proteins. The point at which this behaviour changes has been unambiguously defined enabling the direct comparison of phase transition of proteins during partition in all systems. The results obtained show that phase transition during adsorption and partition occur at similar concentrations of salt. This is less than that required to promote precipitation. It appears, from these limited studies, that top-phase preferring proteins are partitioned at salt concentrations above those required to cause adsorption. Proteins preferring the lower phase are partitioned at salt concentrations close to or below those required for adsorption. This raises questions regarding the solvated molecular form of the partitioned proteins and the definition of the partition coefficient.     

Temperature dependence of the NMR generalized order parameter

The study of protein conformational dynamics is motivated in large part by a desire to understand the forces present at different sites throughout the molecular structure. The generalized order parameter determined by NMR spectroscopy has played a central role in the study of protein dynamics on the picosecond-nanosecond time scale. A modeling procedure is presented for analysis of the temperature dependence of the generalized order parameter that extends a previous analysis (Massi and Palmer, J Am Chem Soc 2003;125:11158-11159). As part of this procedure, the potential of mean force is characterized for the N-H bond vectors of the protein backbone. This procedure accounts for the observed temperature dependence of the generalized order parameter in a representative data set from the B1 domain of Streptococcal protein G (Seewald, Pichumani, Stowell, Tibbals, Regan, and Stone, Protein Sci 2000;9:1177-1193). The results indicate a general trend, in which the force constants associated with the potential of mean force decrease with increasing temperature. The analysis also provides evidence for variations in the potential of mean force for different secondary structural elements.     

Hydrophobic forces between protein molecules in aqueous solutions of concentrated electrolyte

Protein-protein interactions have been measured for a mutant (D101F) lysozyme and for native lysozyme in concentrated solutions of ammonium sulfate at pH 7 and sodium chloride at pH 4.5. In the mutant lysozyme, a surface aspartate residue has been replaced with a hydrophobic phenylalanine residue. The protein-protein interactions of D101F lysozyme are more attractive than those of native lysozyme for all conditions studied. The salt-induced attraction is correlated with a solvation potential of mean force given by the work required to desolvate the part of the protein surfaces that is buried by the protein-protein interaction. This work is proportional to the aqueous surface-tension increment of the salt and the fractional non-polar surface coverage of the protein. Experimental measurements of osmotic second virial coefficients validate a proposed potential of mean force that ascribes the salt-induced attraction between protein molecules to an enhancement of the hydrophobic attraction. This model provides a first approximation for predicting the protein-protein potential of mean force in concentrated aqueous electrolyte solutions; this potential is useful for determining solution conditions favorable for protein crystallization.     

Interactions of proteins in aqueous electrolyte solutions from fluorescence anisotropy and circular-dichroism measurements

Understanding aqueous protein-protein interactions is crucial for the development of a molecular-thermodynamic model for salt-induced protein precipitation. In addition, protein interactions are important in many disease states, including cataract formation and alpha-amyloid diseases. Fluorescence anisotropy provides a means to measure intermolecular interactions. In this work, monomer-dimer equilibrium of the peptide T4 LYS(11-36) was studied by fluorescence anisotropy over the pH range 4-7 and the NaCl concentration range 0.0-1.0 M, in a 25 mM sodium phosphate buffer. This 26 amino-acid peptide is derived from the beta-sheet region of the T4 lysozyme molecule and has the potential to form amyloid fibrils. The association constant for dimerization increases with rising pH and ionic strength. The potential of mean force for peptide-peptide interactions was calculated from these association constants. Circular-dichroism measurements show that the peptide becomes more structured as the pH rises, possibly contributing to increased association.     

Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

Partitioning of beta-galactosidase fusion proteins in PEG/potassium phosphate aqueous two-phase systems.

Four different beta-galactosidase fusion proteins have been partitioned in poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase systems. The partition coefficients (K) of staphylococcal protein A-beta-galactosidase (SpA beta gal) (K = 3.5) and staphylococcal protein A-streptococcal protein G-beta-galactosidase (AG beta gal) (K = 2.8) were compared with the partition coefficients of their constituent molecules, beta-galactosidase, SpA, and protein AG. It was found that by fusing beta-galactosidase to the smaller proteins SpA and protein AG, their partition coefficients were increased four to five times. Experimental data were fitted into, and found to agree with, the Albertsson partition model of interacting molecules. The compatibility with PEG and potassium phosphate of beta-galactosidase, SpA, and two different versions of the SpA beta gal protein, displayed as precipitation curves, showed a relationship to the protein partition coefficients in PEG/potassium phosphate systems. High solubility in one phase component was accompanied by preferential partitioning to the phase rich in the same component in the PEG/potassium phosphate system. Also, a changed linker region in SpA beta gal resulted in a more soluble protein. This, together with the improved K values of the target proteins by fusion, shows that it is possible to use beta-galactosidase as an affinity handle.     

Lipid bilayer deformation and the free energy of interaction of a Kv channel gating-modifier toxin

A number of membrane proteins act via binding at the water/lipid bilayer interface. An important example of such proteins is provided by the gating-modifier toxins that act on voltage-gated potassium (Kv) channels. They are thought to partition to the headgroup region of lipid bilayers, and so provide a good system for probing the nature of interactions of a protein with the water/bilayer interface. We used coarse-grained molecular dynamics simulations to compute the one-dimensional potential of mean force (i.e., free energy) profile that governs the interaction between a Kv channel gating-modifier toxin (VSTx1) and model phospholipid bilayers. The reaction coordinate sampled corresponds to the position of the toxin along the bilayer normal. The course-grained representation of the protein and lipids enabled us to explore extended time periods, revealing aspects of toxin/bilayer dynamics and energetics that would be difficult to observe on the timescales currently afforded by atomistic molecular dynamics simulations. In particular, we show for this model system that the bilayer deforms as it interacts with the toxin, and that such deformations perturb the free energy profile. Bilayer deformation therefore adds an additional layer of complexity to be addressed in investigations of membrane/protein systems. In particular, one should allow for local deformations that may arise due to the spatial array of charged and hydrophobic elements of an interfacially located membrane protein.     

Distance-dependent atomic knowledge-based force in protein fold recognition

We have recently introduced a novel model for discriminating the correctly folded proteins from well-designed decoy structures using mechanical interatomic forces. In the model, we considered a protein as a collection of springs and the force imposed to each atom was calculated by using the relation between the potential energy and the force. A mean force potential function is obtained from statistical contact preferences within the known protein structures. In this article, the interatomic forces are calculated by numerical derivation of the potential function. For assessing the knowledge-based force function we consider an optimal structure and define a score function on the 3D structure of a protein. We compare the force imposed to each atom of a protein with the corresponding atom in the optimum structure. Afterwards we assign larger scores to those atoms with the lower forces. The total score is the sum of partial scores of atoms. The optimal structure is assumed to be the one with the highest score in the dataset. Finally, several decoy sets are applied in order to evaluate the performance of our model.     

Shape changes of the human red cell studied by aqueous two-phase partition

Aqueous two-phase systems have been used to study the human red cell during metabolically induced shape changes. When the discoid character of the cells was lost in favour of echinocytic forms, the partition increased both in charge-sensitive and in charge-insensitive two-phase systems. Reversal of the shape transformation by ATP repletion not only led to shape recovery but also restored the initial partition. Therefore it is apparent that red cells exhibit a shape-dependent partition behaviour. As the partition is dependent on surface properties (such as charge and hydrophobicity) of the partitioned material, the results show that the shape changes caused rearrangement of the membrane and thereby exposure of or greater accessibility of binding groups on the cell surface. The similar partition behaviour in the charge-sensitive and charge-insensitive phase systems show that the increased partition was caused mainly by increased hydrophobic interactions between the cells and the upper phase. The observed partition behaviour therefore suggests that the echinocytic cells acquire a higher affinity for the upper phase by repacking the lipid bilayer or at least the outer leaflet into a less efficient packed and thus more fluid structure.     

Quasi-harmonic method for studying very low frequency modes in proteins

A quasi-harmonic approximation is described for studying very low frequency vibrations and flexible paths in proteins. The force constants of the empirical potential function are quadratic approximations to the potentials of mean force; they are evaluated from a molecular dynamics simulation of a protein based on a detailed anharmonic potential. The method is used to identify very low frequency (～1 cm^?1) normal modes for the protein pancreatic trypsin inhibitor. A simplified model for the protein is used, for which each residue is represented by a single interaction center. The quasi-harmonic force constants of the virtual internal coordinates are evaluated and the normal-mode frequencies and eigenvectors are obtained. Conformations corresponding to distortions along selected low-frequency modes are analyzed.     

黑河中游湿地景观破碎化过程及其驱动力分析

在遥感和GIS技术支持下,基于1975-2010年长时间序列遥感影像,选取斑块密度指数(PD)、景观内部生境面积指数(IA)、斑块平均面积指数(MPS)、斑块形状破碎化指数(FS1、FS2)等具有典型生态意义的景观指数模型,系统分析了黑河中游湿地景观的破碎化过程,并结合灰色关联分析、主成分分析等方法,探讨了影响研究区湿地景观破碎化过程的各驱动因子.结果表明:近35年来,研究区湿地景观破碎化主要表现为斑块平均面积的萎缩,斑块密度的上升以及斑块形状破碎化指数的增大.整个研究时段内,研究区湿地斑块平均面积减少了48.95hm2,斑块密度的上升0.006个/hm2;导致黑河中游湿地景观破碎化发生和发展的驱动力包含自然和人文两个方面.自然因子对湿地景观破碎化进程的影响则主要体现在气温和降水上,而且气温对湿地景观破碎化进程的影响程度明显大于降水.但在1975-2010年间的这一较小时间尺度上,人类活动对湿地景观破碎化的贡献率明显高于自然因子,人类活动能力的增强以及影响范围的不断扩大是引发黑河中游湿地景观破碎化的主因.     

The non-polar solvent potential of mean force for the dimerization of alanine dipeptide: the role of solute-solvent van der Waals interactions

The non-polar component of the potential of mean force of dimerization of alanine dipeptide has been calculated in explicit solvent by free energy perturbation. We observe that the calculated PMF is inconsistent with a non-polar hydration free energy model based solely on the solute surface area. The non-linear behavior of the solute-solvent van der Waals energy is primarily responsible for the non-linear dependence of the potential of mean force with respect to the surface area. The calculated potential of mean force is reproduced by an implicit solvent model based on a solvent continuum model for the solute-solvent van der Waals interaction energy and the surface area for the work of forming the solute cavity.     

Use of a potential of mean force to analyze free energy contributions in protein folding.

A method for calculation of the free energy of residues as a function of residue burial is proposed. The method is based on the potential of mean force, with a reaction coordinate expressed by residue burial. Residue burials are calculated from high-resolution protein structures. The largest individual contributions to the free energy of a residue are found to be due to the hydrophobic interactions of the nonpolar atoms, interactions of the main chain polar atoms, and interactions of the charged groups of residues Arg and Lys. The contribution to the free energy of folding due to the uncharged side chain polar atoms is small. The contribution to the free energy of folding due to the main chain polar atoms is favorable for partially buried residues and less favorable or unfavorable for fully buried residues. Comparison of the accessible surface areas of proteins and model spheres shows that proteins deviate considerably from a spherical shape and that the deviations increase with the size of a protein. The implications of these results for protein folding are also discussed.     

Hysteresis and Statistical Errors in Free Energy Perturbation L to D Amino Acid Conversion

Abstract

A theory based on a Langevin equation along the reaction coordinate is developed to explain and calculate systematic and statistical errors in free energy perturbation simulations. The errors are calculated exactly when both the perturbation potential and the mean potential from the surrounding degrees of freedom are harmonic in the reaction coordinate. The effect of the mean potential is small as long as the force constant is small compared to the force constant of the perturbation potential. This indicates that the results obtained with zero mean force may still be valid as long as the second derivate of the mean potential is small compared to that of the perturbation potential. The theory is applied to conversion between L and D amino acids by changing the position of the minimum of the harmonic improper dihedral potential between ±35.264 degrees. For phenylalanine bound in the active site of a protein (thermolysin) we find from 20 psec. simulations statistical errors and hysteresis that both are about 2.5 kJ/mol in agreement with what is obtained from the theoretical predictions. The statistical errors are proportional to the square root of the coupling to the heat bath and inversely proportional to the square root of integration time while the (positive) hysteresis due to that the reaction coordinate lags behind is linear in the same quantities. This shows that the systematic errors will dominate in short simulations while the statistical ones will dominate for long simulations. The treatment is based on that the systematic influence of the surroundings can be represented by a mean force upon the reaction coordinate. If the relaxation processes of the environment are slow this may not be true. Then additional errors have to be considered.     

A pairwise residue contact area-based mean force potential for discrimination of native protein structure

Background  

Considering energy function to detect a correct protein fold from incorrect ones is very important for protein structure prediction and protein folding. Knowledge-based mean force potentials are certainly the most popular type of interaction function for protein threading. They are derived from statistical analyses of interacting groups in experimentally determined protein structures. These potentials are developed at the atom or the amino acid level. Based on orientation dependent contact area, a new type of knowledge-based mean force potential has been developed.     

Scanning molecular sieve chromatography of interacting protein systems. II. Determination of large zone transport parameters by the difference profile method at low solute concentration

The experimental determination of difference profiles for the study of large zone transport processes by scanning molecular sieve chromatography is described. Using the difference profile method, the progesterone-induced purple glycoprotein of the porcine uterus was found to exist as monomeric units in high ionic environment, with a partition coefficient of 0.269, partition cross-section of 0.488, partition radius of 25 A and a molecular weight of 33500 g mole . The technique was further applied in examining the association-dissociation properties of oxyhemoglobin. In a high tonic environment, the partition coefficient was found to be 0.365 for dimer and the partition cross-section, 0.419; for the tetramer in low ionic strength solution, the partition coefficient was 0.275 and the partition cross-section 0.377, with a dissociation constant of 1.03 x 10(-6) mole/1. This new technique should prove applicable in (1) readily locating the centroid positions of transport boundary profiles at the lowest practicable protein concentration limits, (2) demonstrating the characteristic boundary shape and concentration-dependent centroid position for an interacting solute, (3) determining the axial dispersion coefficient characteristic of solute turbulence within the gel matrix, and (4) distinguishing the boundary between low and high ionic strength solvent phases in the gel column.     

Scanning molecular sieve chromatography of interacting protein systems. II. Determination of large zone transport parameters by the difference profile method at low solute concentration.

The experimental determination of difference profiles for the study of large zone transport processes by scanning molecular sieve chromatography is described. Using the difference profile method, the progesterone-induced purple glycoprotein of the porcine uterus was found to exist as monomeric units in high ionic environment, with a partition coefficient of 0.269, partition cross-section of 0.488, partition radius of 25 A and a molecular weight of 33,500 g/mole. The technique was further applied in examining the association-dissociation properties of oxyhemoglobin. In a high tonic environment, the partition coefficient was found to be 0.365 for dimer and the partition cross-section, 0.419; for the tetramer in low ionic strength solution, the partition coefficient was 0.275 and the partition cross-section 0.377, with a dissociation constant of 1.03 x 10(-6) mole/l. This new technique should prove applicable in (1) readily locating the centroid positions of transport boundary profiles at the lowest practible protein concentration limits, (2) demonstrating the characteristic boundary shape and concentration-dependent centroid position for an interacting solute, (3) determining the axial dispersion coefficient characteristic of solute turbulence within the gel matrix, and (4) distinguishing the boundary between low and high ionic strength solvent phases in the gel column.     

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.     

Reticulon short hairpin transmembrane domains are used to shape ER tubules

Reticulons are integral membrane proteins that partition into and shape the tubular endoplasmic reticulum (ER). We propose that reticulons use a membrane insertion mechanism to generate regions of high membrane curvature in the ER. A reticulon contains two short hairpin transmembrane domains (TMDs), which could generate membrane curvature by increasing the area of the cytoplasmic leaflet. Here, we test whether the short length of these hairpin TMDs is required for reticulon membrane-shaping functions in mammalian cells. We lengthened the TMDs of reticulon 4 to resemble a typical bi-pass TMD that spans both leaflets. We find that TMD mutants oligomerize like wild type (wt), however, they are not immobilized, do not partition into tubules, do not constrict tubules and no longer suppress peripheral ER cisternae. Therefore, short hairpin TMD length is required for reticulon protein partitioning and membrane-shaping functions. Another membrane protein with a short hairpin TMD is caveolin. We show that an ER-retained caveolin construct also partitions within the ER in a manner that is dependent on it containing a short hairpin TMD. These data suggest that a short hairpin TMD may be a general feature used by membrane-shaping proteins to partition into and shape regions of high membrane curvature.