Role Of Salt Bridges Research Articles

A computational study on heat flow and thermostability of thermophilic and mesophilic proteins

Thermophilic and mesophilic proteins exhibit distinct responses to temperature variations, a critical aspect of their biological functionality. Understanding the microscopic mechanisms governing protein stability and flexibility at different temperatures is essential. Vibrational energy transfer within proteins plays a pivotal role in their diverse biological processes. We employed vibrational energy diffusivity methodology to investigate this phenomenon. Molecular dynamics simulations were conducted at multiple temperatures such as 308, 325, and 350 K, revealing that fluctuations persist in both thermophilic and mesophilic proteins. To delve deeper into the system's stability, we constructed frequency-resolved communication maps, highlighting key residues involved in energy transfer pathways. Our results identify highly active residues that influence protein stabilization or destabilization. Furthermore, the role of salt bridges and non-covalent interactions in protein stability is explored. Our findings offer insights into the relationship between protein activity and thermal resistance, contributing to a comprehensive understanding of thermophilic protein's behavior at different temperatures. This study provides valuable insights into the protein's flexibility and stability, shedding light on the mechanisms that underpin their biological functions.

A role of salt bridges in mediating drug potency: A lesson from the N-myristoyltransferase inhibitors.

The salt bridge is the strongest non-covalent interaction in nature and is known to participate in protein folding, protein-protein interactions, and molecular recognition. However, the role of salt bridges in the context of drug design has remained not well understood. Here, we report that a common feature in the mechanism of inhibition of the N-myristoyltransferases (NMT), promising targets for the treatment of protozoan infections and cancer, is the formation of a salt bridge between a positively charged chemical group of the small molecule and the negatively charged C-terminus of the enzyme. Substituting the inhibitor positively charged amine group with a neutral methylene group prevents the formation of the salt bridge and leads to a dramatic activity loss. Molecular dynamics simulations have revealed that salt bridges stabilize the NMT-ligand complexes by functioning as molecular clips that stabilize the conformation of the protein structure. As such, the creation of salt bridges between the ligands and their protein targets may find an application as a valuable tool in rational drug design.

Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order, and molecular chaperone activity

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer-monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.

The role of salt bridges on the temperature adaptation of aqualysin I, a thermostable subtilisin-like proteinase

Differences in salt bridges are believed to be a structural hallmark of homologous enzymes from differently temperature-adapted organisms. Nevertheless, the role of salt bridges on structural stability is still controversial. While it is clear that most buried salt bridges can have a functional or structural role, the same cannot be firmly stated for ion pairs that are exposed on the protein surface. Salt bridges, found in X-ray structures, may not be stably formed in solution as a result of high flexibility or high desolvation penalty. More studies are thus needed to clarify the picture on salt bridges and temperature adaptation. We contribute here to this scenario by combining atomistic simulations and experimental mutagenesis of eight mutant variants of aqualysin I, a thermophilic subtilisin-like proteinase, in which the residues involved in salt bridges and not conserved in a psychrophilic homolog were systematically mutated. We evaluated the effects of those mutations on thermal stability and on the kinetic parameters.Overall, we show here that only few key charged residues involved in salt bridges really contribute to the enzyme thermal stability. This is especially true when they are organized in networks, as here attested by the D17N mutation, which has the most remarkable effect on stability. Other mutations had smaller effects on the properties of the enzyme indicating that most of the isolated salt bridges are not a distinctive trait related to the enhanced thermal stability of the thermophilic subtilase.

Transmembrane helix assembly and the role of salt bridges.

Transmembrane helix-helix interactions mediate the folding and assembly of membrane proteins. Recognition motifs range from GxxxG and leucine zippers to polar side chains and salt bridges. Some canonical membrane proteins contain local charge clusters that are important for folding and function, and which have to be compatible with a stable insertion into the bilayer via the translocon. Recently, the electrostatic "charge zipper" has been described as another kind of assembly motif. The protein sequences exhibit a quasi-symmetrical pattern of complementary charges that can form extended ladders of salt bridges. Such segments can insert reversibly into membranes, or even translocate across them. Nature uses charge zippers in transport processes, and they can also be adapted in the design of cell-penetrating carriers.

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.

The Role of Salt Bridges, Charge Density, and Subunit Flexibility in Determining Disassembly Routes of Protein Complexes

SummaryMass spectrometry can be used to characterize multiprotein complexes, defining their subunit stoichiometry and composition following solution disruption and collision-induced dissociation (CID). While CID of protein complexes in the gas phase typically results in the dissociation of unfolded subunits, a second atypical route is possible wherein compact subunits or subcomplexes are ejected without unfolding. Because tertiary structure and subunit interactions may be retained, this is the preferred route for structural investigations. How can we influence which pathway is adopted? By studying properties of a series of homomeric and heteromeric protein complexes and varying their overall charge in solution, we found that low subunit flexibility, higher charge densities, fewer salt bridges, and smaller interfaces are likely to be involved in promoting dissociation routes without unfolding. Manipulating the charge on a protein complex therefore enables us to direct dissociation through structurally informative pathways that mimic those followed in solution.

Effects of High Temperature on Desolvation Costs of Salt Bridges across Protein Binding Interfaces: Similarities and Differences between Implicit and Explicit Solvent Models

The role of salt bridges in protein-protein binding is largely determined by the costs of desolvating the oppositely charged members of the salt bridge upon binding. On the basis of Poisson-Boltzmann (PB) implicit solvent calculations, it has been proposed that the reduced desolvation penalties of salt bridges at high temperatures provide one explanation for the increased abundance of salt bridges in hyperthermophilic proteins. Here, for the first time, we directly compare the PB implicit solvent model with several explicit water models in computing the effects of extremely high temperature (i.e., 100 °C) on the desolvation penalties of salt bridges across protein-protein interfaces. With the exception of two outliers, the desolvation costs at 100 °C from implicit and explicit solvent calculations are of similar magnitudes and significantly reduced relative to 25 °C. The two outliers correspond to salt bridges that are both buried and part of a salt bridge network, a challenging case that should be considered in the development of fast solvation models.

Role of Hydrophobic Core on the Thermal Stability of Proteins—Molecular Dynamics Simulations on a Single Point Mutant of Sso7d

The role of salt bridges in chromatin protein Sso7d, from S. solfataricus has previously been shown to be crucial for its unusual high thermal stability. Experimental studies have shown that single site mutation of Sso7d (F31A) leads to a substantial decrease in the thermal stability of this protein due to distortion of the hydrophobic core. In the present study, we have performed a total of 0.2 μs long molecular dynamics (MD) simulations on F31A at room temperature, and at 360 K, close to the melting temperature of the wild type (WT) protein to investigate the role of hydrophobic core on protein stability. Sso7d-WT was shown to be stable at both 300 and 360 K; however, F31A undergoes denaturation at 360 K, consistent with experimental results. The structural and energetic properties obtained using the analysis of MD trajectories indicate that the single mutation results in high flexibility of the protein, and loosening of intramolecular interactions. Correlation between the dynamics of the salt bridges with the structural transitions and the unfolding pathway indicate the importance of both salt bridges and hydrophobic in effecting thermal stability of proteins in general.

Effects of High Temperature on Desolvation Costs of Salt Bridges Across Protein Binding Interfaces: Similarities and Differences between Implicit and Explicit Solvent Models

The role of salt bridges in protein-protein binding is largely determined by the costs of desolvating the oppositely charged members of the salt bridge upon binding. On the basis of Poisson-Boltzmann (PB) implicit solvent calculations, it has been proposed that the reduced desolvation penalties of salt bridges at high temperatures provide one explanation for the increased abundance of salt bridges in hyperthermophilic proteins. Here, for the first time, we directly compare the PB implicit solvent model with several explicit water models in computing the effects of extremely high temperature (i.e., 100 °C) on the desolvation penalties of salt bridges across protein-protein interfaces. With the exception of two outliers, the desolvation costs at 100 °C from implicit and explicit solvent calculations are of similar magnitudes and significantly reduced relative to 25 °C. The two outliers correspond to salt bridges that are both buried and part of a salt bridge network, a challenging case that should be considered in the development of fast solvation models.

Conformational flexibility, binding energy, role of salt bridge and alanine-mutagenesis for c-Abl kinase complex

Abl kinase plays a decisive role in the mechanism of the most fatal human pathogen chronic mylogenous leukemia (CML). Here, we have carried out a comprehensive study about the conformational flexibility, role of salt bridge and the protein- ligand interaction for this kinase with its well-known inhibitor, Imatinib. We have performed molecular dynamics simulations for conformational behavior, investigated the salt bridges and calculated the binding free energy of Imatinib with MM-PB/SA method for Abl kinase complex. We also explored the role of salt-bridge in the kinase complex and its effect on binding activity of inhibitors. Furthermore, to investigate the importance of those residues which form salt bridges, we mutated them by Alanine with the help of Alanine scanning program. We noticed significant variations in total free energy of Imatinib in all possible mutations. The binding free energy of ligand for kinase receptor was analyzed by molecular mechanics Poission Boltzmann surface area (MM-PB/SA) method. These results suggest that conserved glutamic acid and lysine are necessary for stability of complex.

Role of Salt Bridges within the Ligand Binding Domains of NMDA Receptors During Receptor Activation

Glutamatergic synaptic transmission mediated by the N-methyl-D-aspartate (NMDA) receptor plays a fundamental role in numerous central nervous system processes, but how the receptor becomes active is insufficiently understood. Structural studies of isolated ligand-binding domains (LBDs) revealed that agonists bind between two flexible lobes (D1 and D2) and by causing the lobes to move closer together initiate the activation reaction. In many cases, the efficacy of the agonist correlates with how narrow the cleft becomes and/or how stable the closed-cleft conformation is. In NMDA receptors, full-agonists induce cleft-closure and the formation of new stabilizing electrostatic interactions across the lobes of soluble LBDs. However, how these interactions contribute to the kinetics of NMDA receptors is not known. To evaluate the roles of putative cross-cleft interactions to the activation mechanism of intact receptors, we eliminated these salt bridges in individual subunits and quantified their effect on gating by kinetic analyses of single channel traces. We were surprised to find that: receptors containing GluN1(K483A) and GluN2A(K487A) had activities that were ∼10% higher than wild-type controls; receptors containing GluN1(K483M) had wild-type activity; and those containing GluN2(N687L) had activities that were ∼30% lower than controls. These results suggest that at these positions, residue-size as well as the ability to form cross-cleft interactions affect the gating process. They also reveal subtle distinctions between how residues in the GluN1 and GluN2A LBDs contribute to gating.

Apolipoprotein E structural requirements for the formation of SDS-stable complexes with beta-amyloid-(1-40): the role of salt bridges.

Of the three major isoforms of human apolipoprotein E (apoE), apoE4 is a risk factor for the development of Alzheimer's disease. Among possible neurologically relevant differences in the properties of apoE3 and apoE4 is the fact that apoE3 forms an SDS-stable complex with beta-amyloid-(1-40) (Abeta40) with greater avidity than does apoE4. This interaction may sequester potentially toxic species of Abeta or facilitate clearance. To understand more about this difference, we examined whether differences in salt bridges between apoE domains influence the capacity of apoE isoforms to form complexes with Abeta. In apoE3 there is a salt bridge between Arg-61 and Asp-65, while in apoE4 there are salt bridges between Arg-61 and Glu-255, and Arg-112 and Glu-109. Mutation of position 112, which is Cys in apoE3 and Arg in apoE4, to Ala or Lys abolished complex formation, while mutant apoE with Ser at this position retained the capacity to form complex. Substituting Ala for Glu-109 had no effect on the ability of either apoE4 or apoE3 to form complexes. On the other hand, substitution of Thr for Arg-61 in apoE3 abolished, and truncation of apoE3 at position 201 substantially lowered, but did not abolish, complex formation. Neither of these mutations within apoE4 had any affect on its complex formation with Abeta. These results suggest that the nature of the cysteine residue in apoE3 and interactions between the N-terminal and C-terminal domains of human apoE are important for the ability of apoE3 to form an SDS-stable complex with Abeta40.

The Bohr Effect of Hemoglobin Intermediates and the Role of Salt Bridges in the Tertiary/Quaternary Transitions

Understanding mechanisms in cooperative proteins requires the analysis of the intermediate ligation states. The release of hydrogen ions at the intermediate states of native and chemically modified hemoglobin, known as the Bohr effect, is an indicator of the protein tertiary/quaternary transitions, useful for testing models of cooperativity. The Bohr effects due to ligation of one subunit of a dimer and two subunits across the dimer interface are not additive. The reductions of the Bohr effect due to the chemical modification of a Bohr group of one and two alpha or beta subunits are additive. The Bohr effects of monoliganded chemically modified hemoglobins indicate the additivity of the effects of ligation and chemical modification with the possible exception of ligation and chemical modification of the alpha subunits. These observations suggest that ligation of a subunit brings about a tertiary structure change of hemoglobin in the T quaternary structure, which breaks some salt bridges, releases hydrogen ions, and is signaled across the dimer interface in such a way that ligation of a second subunit in the adjacent dimer promotes the switch from the T to the R quaternary structure. The rupture of the salt bridges per se does not drive the transition.

3P011超好熱菌トリプトファン合成酵素αサブユニットの安定化に果たすイオン結合の役割

3P011超好熱菌トリプトファン合成酵素αサブユニットの安定化に果たすイオン結合の役割

Role of salt bridge(s) in the binding and photoconversion of bilirubin bound to high affinity site on human serum albumin

The role of salt bridge(s) (between ϵ-NH 2 groups of lysine residues of human serum albumin (HSA) and carboxyl groups of bilirubin) in the binding and photoconversion of bilirubin bound to high affinity site on HSA was investigated by covalent modification of ∼20% internal (buried) lysine residues of HSA with acetic anhydride, succinic anhydride and O-methylisourea and white light irradiation of their complexes with bilirubin. The different HSA derivatives, namely, acetylated HSA (aHSA), succinylated HSA (sHSA) and guanidinated HSA (gHSA), thus obtained, were found to be homogeneous with respect to charge and size and characterized in detail in terms of mean residue ellipticity, Stokes radius, tryptophan fluorescence, bilirubin binding and the photochemistry of their complexes with bilirubin. All the three derivatives retained helical contents and molecular size (Stokes radius) similar to HSA except for sHSA which showed a slight increase in the Stokes radius from 3.56 to 3.64 nm. Further, fluorescence properties of aHSA and sHSA were also found to be different from HSA and gHSA. Based on difference spectral change, fluorescence quenching and fluorescence enhancement results of bilirubin bound to HSA and its derivatives, nearly 46 and 48% reduction in bilirubin binding was observed in the case of aHSA and sHSA, respectively. Both aHSA and sHSA showed a decrease of 8- and 10-fold, respectively, in association constant compared to native HSA. Although the bisignate circular dichroism (CD) spectra of an equimolar (1:1) bilirubin–HSA complex was retained by all three HSA derivatives, the intensity of both positive and negative CD Cotton effects decreased significantly in both aHSA and sHSA. gHSA which retained net charge identical to native HSA, showed little decrease in bilirubin binding and the intensity of bisignate CD Cotton effects. The photochemical reaction of bilirubin bound to aHSA and sHSA produced opposite results to those observed with HSA and gHSA. A brief (2 min) irradiation of an equimolar complex of bilirubin with both aHSA and sHSA accompanied a rapid shift (14–15 nm) in the absorption spectrum of the bound pigment towards the blue region and almost complete elimination of negative CD Cotton effects while only moderately affecting the magnitude of positive CD Cotton effects. On the other hand, similar treatment of the complexes of bilirubin with HSA and gHSA did not show any change in the absorption spectrum, only a slight decrease in the intensity of both positive and negative CD Cotton effects was observed. The fluorescence intensity of bilirubin bound to HSA and gHSA was increased upon irradiation with white light and after 30 min it was nearly twice the value observed at 0 min irradiation. Interestingly, no change in the fluorescence intensity of bilirubin bound either to aHSA or sHSA was observed upon irradiation, even on increasing the duration of irradiation to 1 h. Taken together, the results on fluorescence quenching, fluorescence enhancement, CD spectral changes and visible absorption spectroscopy suggest that salt bridge(s) of the type (–COO −.... +H 3N–) in which the ϵ-NH 2 group(s) contributed by lysine residues, are not only involved in the enantioselective binding of bilirubin but also in the stereospecific photoisomerization of bilirubin bound to a high affinity site on HSA.

Gas-phase basicity of (CH3)3N+-C6H4-COO- zwitterions: a new class of organic super bases.

Gas-phase basicity of (CH3)3N+-C6H4-COO- zwitterions: a new class of organic super bases.

Molecular Dynamics on a Model for Nascent High-Density Lipoprotein: Role of Salt Bridges

The results of an all-atom molecular dynamics simulation on a discoidal complex made of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) and a synthetic α-helical 18-mer peptide with an apolipoprotein-like charge distribution are presented. The system consists of 12 acetyl-18A-amide (Ac-18A-NH 2) (Anantharamaiah et al., 1985. J. Biol. Chem. 260:10248–10255) molecules and 20 molecules of POPC in a bilayer, 10 in each leaflet, solvated in a sphere of water for a total of 28,522 atoms. The peptide molecules are oriented with their long axes normal to the bilayer (the “picket fence” orientation). This system is analogous to complexes formed in nascent high-density lipoprotein and to Ac-18A-NH 2/phospholipid complexes observed experimentally. The simulation extended over 700 ps, with the last 493 ps used for analysis. The symmetry of this system allows for averaging over different helices to improve sampling, while maintaining explicit all-atom representation of all peptides. The complex is stable on the simulated time scale. Several possible salt bridges between and within helices were studied. A few salt bridge formations and disruptions were observed. Salt bridges provide specificity in interhelical interactions.

Conservation of Salt Bridges in Protein Families

A detailed computational analysis is presented that focuses on the relationship between structural attributes and the degree and mode of salt bridge conservation. A data set of conserved and non-conserved salt bridges was constructed from eight protein families, based on the structural alignment of family members. Salt bridges were defined at the secondary structure level rather than at the residue level, implying different possible modes of conservation: preservation (same charges at the same residue positions), compensation (reversal of charges), and complementation (maintenance of a salt bridge between two segments of secondary structures, not involving the same residue positions). Structural attributes such as the surface accessibility, distance from the active site, or type of secondary structures involved, were studied. No significant differences were found between conserved and non-conserved salt bridges, except for the surface accessibility. Conserved salt bridges were shown to be less exposed than non-conserved ones. Moreover, within the set of conserved salt bridges, the degree of conservation was shown to negatively correlate with surface exposure; however, not to an extent that could indicate a general role for electrostatic interactions in the protein interior. Examination of the most conserved salt bridge in each family showed a variety of typical features: Some involved the terminal segments of the protein, some were buried and one involved the catalytic site of the protein. Hence, the role of salt bridges is more specific, probably in fine tuning of a specific structure through the folding process or in determining the functional site. As for the conservation mode, preservations were found to predominate in the conserved interactions, while complementations were of secondary importance. Compensations occurred only rarely and mostly in exposed salt bridges, suggesting that this mechanism is not utilized frequently and especially not in important interactions.

Effect of Removal of a Salt-Bridge on the Oxygen Binding Properties and the Electronic Structure of Heme in Cobalt-Iron Hybrid Hemoglobin

In order to clarify the role of salt-bridges in hemoglobin, the oxygen equilibrium curves and electron paramagnetic resonance (EPR) spectra of cobalt-iron hybrid hemoglobins were determined. The EPR spectra of deoxy alpha(Co)2 beta(Fe)2 could be interpreted as a mixture of two distinct paramagnetic species: one showed a maximum of the first derivative spectrum at g = 2.39 and the other at g = 2.33. The oxygen equilibrium curves of the hybrid indicated that the former is assignable to the T structure and the latter to the R structure. The cooperativity of oxygen binding of alpha(Co)2 beta(Fe)2 exhibited a maximum at g = 2.33, which is characteristic of the R structure, regardless of the pH. Addition of inositol hexaphosphate (IHP) to des-Arg alpha(Co)2 beta(Fe)2 restored the cooperativity of oxygen binding, which implies that the deoxygenated form of des-Arg alpha(Co)2 beta(Fe)2 is converted to the T structure upon addition of IHP. However, the EPR signal at g = 2.39 was not restored upon conversion to the T structure by addition of IHP. It is therefore concluded that the EPR spectrum of the deoxy alpha(Co) subunit depends both on the quaternary structure and on the localized strain at the heme.