Energy of ion pairs in proteins

The energy of ions interaction in an ionic pair and the energy of ions transfer from water into protein at different ions disposition relative to the protein/water boundary has been considered. Ultimately, the ions transfer from aqueous phase into protein, i. e. in the medium with a low dielectric constant is energetically unfavorable and hence it cannot stabilize the protein structure by itself. For stabilization of ionic pairs in protein the action of some additional factors is necessary, in particular the action of the intraglobular electric field.     

Analysis of interactions of buried polar side chains in beta-proteins

It was shown in qualitative and quantitative analyses of polar side chains inaccessible for water molecules as well as their interactions in 100 globular beta-structural proteins that completely buried polar side chains are widespread in beta-proteins, their vast majority being involved in "side chain-side chain" or "side chain-main chain" interactions. The analysis of the occurrence of different "side chain-partner" pairs permitted us to demonstrate that such interactions are selective. The results were compared with similar data obtained previously for alpha-helical proteins.     

Fluctuations in ion pairs and their stabilities in proteins

This report investigates the effect of systemic protein conformational flexibility on the contribution of ion pairs to protein stability. Toward this goal, we use all NMR conformer ensembles in the Protein Data Bank (1) that contain at least 40 conformers, (2) whose functional form is monomeric, (3) that are nonredundant, and (4) that are large enough. We find 11 proteins adhering to these criteria. Within these proteins, we identify 22 ion pairs that are close enough to be classified as salt bridges. These are identified in the high-resolution crystal structures of the respective proteins or in the minimized average structures (if the crystal structures are unavailable) or, if both are unavailable, in the "most representative" conformer of each of the ensembles. We next calculate the electrostatic contribution of each such ion pair in each of the conformers in the ensembles. This results in a comprehensive study of 1,201 ion pairs, which allows us to look for consistent trends in their electrostatic contributions to protein stability in large sets of conformers. We find that the contributions of ion pairs vary considerably among the conformers of each protein. The vast majority of the ion pairs interconvert between being stabilizing and destabilizing to the structure at least once in the ensembles. These fluctuations reflect the variabilities in the location of the ion pairing residues and in the geometric orientation of these residues, both with respect to each other, and with respect to other charged groups in the remainder of the protein. The higher crystallographic B-factors for the respective side-chains are consistent with these fluctuations. The major conclusion from this study is that salt bridges observed in crystal structure may break, and new salt bridges may be formed. Hence, the overall stabilizing (or, destabilizing) contribution of an ion pair is conformer population dependent.     

The greater strength of arginine: carboxylate over lysine carboxylate ion pairs implications for the design of novel enzymes and drugs

The rational design of enzyme catalysts for chiral chemistry and of drugs which bind to proteins would be facilitated if rules for the recognition of one partner by the other could be formulated. This communication suggests and tests one generalization: arginine forms a tighter ion pair with a carboxylate group than does lysine and is always used for ion-pairs which are not broken during turnover in naturally-occurring enzymes.     

Inorganic ion pairs in physiology: significance and quantitation

1. Body fluids contain ion pairs such as NaC10, CaCl+, CaHCO+3 and MgSO04. 2. Ion pairs must often be considered in the quantitation of acid-base, solubility, Donnan and other equilibria. 3. Fluxes of ion pairs, such as NaCO-3 and perhaps NaSO-4 and NaCl0, may sometimes contribute significantly to total ion fluxes across cell membranes. 4. Dissociation constants for ion pairs are discussed, but values appropriate to body fluids are often uncertain.     

Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.     

Systematic analysis of buried polar side chains and their interactions in alpha-helical proteins

Examination of 80 alpha-helical proteins and domains demonstrates that they contain from 1 to more than 20 completely buried (water-inaccessible) polar side chains. As a rule the latter have partners for H-bonding but the resulting H-bond system is often not exhaustive. Basing on statistical analysis, we determined the optimal number of H-bonds for every type of polar side chain, and discuss the structural role of vacant donors and acceptors. About half of the H-bonds formed by buried side chains pertain to interhelix contacts of the (side chain)-(side chain) and (side chain)-(main chain) types. Such interactions appear to be a most important factor determining the mutual arrangement of alpha-helices in proteins. Analysis of the frequency of occurrence of various interacting pairs reveals that these interactions are selective.     

Activities of digestive enzymes of polar and subtropical echinoderms

Activities and characteristics of digestive enzymes of polar and subtropical echinoderms are similar. Specific adaptations to facilitate digestion at low temperature were not observed. Levels of chymotrypsin and β-glucosidase of digestive tissues of the Antarctic asteroids Odontaster validus and Odontaster meridionalis, and the Antarctic echinoid Sterechinus neumayeri, are comparable to levels of the asteroids Henricia downii and Astropecten articulatus from the Gulf of Mexico. α-Glucosidase activities are similar for S. neumayeri and A. articulatus. The pH of maximal activity is 7.5 for disaccharidases and 8.5–9.5 for chymotrypsin for both polar and subtropical species. Affinities for substrates were higher at 25°C than at 0°C for disaccharidases of all species. However, K_m values for chymotrypsin increased from 0 to 25°C. Lack of sufficient adaptation by polar echinoderms to facilitate digestion at low temperature may reduce their capacity to digest food. Received: 24 July 1996 / Accepted: 31 January 1997     

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.     

Modeling hydration mechanisms of enzymes in nonpolar and polar organic solvents

A comprehensive study of the hydration mechanism of an enzyme in nonaqueous media was done using molecular dynamics simulations in five organic solvents with different polarities, namely, hexane, 3-pentanone, diisopropyl ether, ethanol, and acetonitrile. In these solvents, the serine protease cutinase from Fusarium solani pisi was increasingly hydrated with 12 different hydration levels ranging from 5% to 100% (w/w) (weight of water/weight of protein). The ability of organic solvents to 'strip off' water from the enzyme surface was clearly dependent on the nature of the organic solvent. The rmsd of the enzyme from the crystal structure was shown to be lower at specific hydration levels, depending on the organic solvent used. It was also shown that organic solvents determine the structure and dynamics of water at the enzyme surface. Nonpolar solvents enhance the formation of large clusters of water that are tightly bound to the enzyme, whereas water in polar organic solvents is fragmented in small clusters loosely bound to the enzyme surface. Ions seem to play an important role in the stabilization of exposed charged residues, mainly at low hydration levels. A common feature is found for the preferential localization of water molecules at particular regions of the enzyme surface in all organic solvents: water seems to be localized at equivalent regions of the enzyme surface independently of the organic solvent employed.     

Recognition of base pairs by polar peptides in double stranded DNA.

The resonance Raman spectrum of native DNA has been obtained using excitation at 257 nm. In a first part, the spectral lines are assigned to the different nucleotide bases which provide the resonance effect. In a second part, the interactions of DNA with basic peptides (Arginine Methylester, Lysine Methylester, Arginyl-Arginine) are investigated using excitation at 300 nm and 257 nm, which give complementary information about the DNA. Both Arginine Methylester and Arginyl-arginine recognize the A-T base pairs, the first one in the large groove, the second one in the narrow groove of DNA. The DNA-Lysine Methylester interaction is very likely not specific but can take place in the large groove of DNA.     

Primary radical ion pairs in photosystem II core complexes

Ultrafast absorption spectroscopy with 20-fs resolution was applied to study primary charge separation in spinach photosystem II (PSII) reaction center (RC) and PSII core complex (RC complex with integral antenna) upon excitation at maximum wavelength 700–710 nm at 278 K. It was found that the initial charge separation between P680* and Chl_D1 (Chl-670) takes place with a time constant of ～1 ps with the formation of the primary charge-separated state P680* with an admixture of: P680^*(1?δ) (P680^δ+1Chl _D1 ^δ? ), where δ ～ 0.5. The subsequent electron transfer from P680^δ+Chl _D1 ^δ? to pheophytin (Pheo) occurs within 13 ps and is accompanied by a relaxation of the absorption band at 670 nm (Chl _D1 ^δ? ) and bleaching of the Pheo_D1 bands at 420, 545, and 680 nm with development of the Pheoband at 460 nm. Further electron transfer to Q_A occurs within 250 ps in accordance with earlier data. The spectra of P680⁺ and Pheo^? formation include a bleaching band at 670 nm; this indicates that Chl-670 is an intermediate between P680 and Pheo. Stimulated emission kinetics at 685 nm demonstrate the existence of two decaying components with time constants of ～1 and ～13 ps due to the formation of P680^δ+Chl _D1 ^δ? and P680⁺Pheo _D1 ^? , respectively.     

Voltage sensor of ion channels and enzymes

Placed in the cell membrane (a two-dimensional environment), ion channels and enzymes are able to sense voltage. How these proteins are able to detect the difference in the voltage across membranes has attracted much attention, and at times, heated debate during the last few years. Sodium, Ca²⁺ and K⁺ voltage-dependent channels have a conserved positively charged transmembrane (S4) segment that moves in response to changes in membrane voltage. In voltage-dependent channels, S4 forms part of a domain that crystallizes as a well-defined structure consisting of the first four transmembrane (S1–S4) segments of the channel-forming protein, which is defined as the voltage sensor domain (VSD). The VSD is tied to a pore domain and VSD movements are allosterically coupled to the pore opening to various degrees, depending on the type of channel. How many charges are moved during channel activation, how much they move, and which are the molecular determinants that mediate the electromechanical coupling between the VSD and the pore domains are some of the questions that we discuss here. The VSD can function, however, as a bona fide proton channel itself, and, furthermore, the VSD can also be a functional part of a voltage-dependent phosphatase.     

Simultaneous siRNA-mediated silencing of pairs of genes coding for enzymes involved in glycosaminoglycan synthesis

It has been demonstrated recently that it is possible to decrease expression of genes coding for enzymes involved in synthesis of glycosaminoglycans (GAGs) by using specific siRNAs which interfere with stability of particular mRNAs. This procedure has been proposed as a potential treatment for patients suffering from mucopolysaccharidoses, a group of inherited metabolic diseases caused by dysfunction of enzymes required for GAG degradation, and resultant storage of these compounds in cells of affected persons. Here, we asked if the simultaneous use two species of specific siRNAs aimed at silencing two genes involved in particular steps of GAG synthesis may be more effective than the use of single siRNA. We found that inhibition of GAG synthesis in cells treated with two siRNAs is generally more effective than using single siRNAs. However, the differences were not statistically significant, therefore the potential benefit from the use of two siRNAs over the use of a single siRNA is doubtful in the light of the cost-benefit ratio and possibly stronger side-effects of the putative therapy.     

Role of water molecules and ion pairs in Dps and related ferritin-like structures

A comparative study of water molecules and ion pairs in 11 Dps protein structures has been carried out. The invariant and common water molecules, the conserved residues interacting with them and the conserved ion pairs have been analyzed. Certain water molecules found on the interfaces between subunits are highly conserved and may be implicated in flexibility or continuing association of the subunits of the structure. It is possible that the water molecules, ion pairs and the special case of a water mediated charged network through a single water molecule are involved in maintaining the stability of the protein.     

Putative interhelix ion pairs involved in the stability of myoglobin.

An earlier theoretical study predicted that specific ion pair interactions between neighboring helices should be important in stabilizing myoglobin. To measure these interactions in sperm whale myoglobin, single mutations were made to disrupt them. To obtain reliable DeltaG values, conditions were found in which the urea induced unfolding of holomyoglobin is reversible and two-state. The cyanomet form of myoglobin satisfies this condition at pH 5, 25 degrees C. The unfolding curves monitored by far-UV CD and Soret absorbance are superimposable and reversible. None of the putative ion pairs studied here makes a large contribution to the stability of native myoglobin. The protein stability does decrease somewhat between 0 and 0.1 M NaCl, however, indicating that electrostatic interactions contribute favorably to myoglobin stability at pH 5.0. A previous mutational study indicated that the net positive charge of the A[B]GH subdomain of myoglobin is an important factor affecting the stability of the pH 4 folding intermediate and potential ion pairs within the subdomain do not contribute significantly to its stability. One of the assumptions made in that study is tested here: replacement of either positively or negatively charged residues outside the A[B]GH subdomain has no significant effect on the stability of the pH 4 molten globule.