Network Of Electrostatic Interactions Research Articles

Freeze-Drying of Proteins in Glass Solids Formed by Basic Amino Acids and Dicarboxylic Acids

The purpose of this study was to produce and characterize glass-state amorphous solids containing amino acids and organic acids that protect co-lyophilized proteins. Thermal analysis of frozen solutions containing a basic amino acid (e.g., L-arginine, L-lysine, L-histidine) and a hydroxy di- or tricarboxylic acid (e.g., citric acid, L-tartaric acid, DL-malic acid) showed glass transition of maximally freeze-concentrated solute at temperatures (T'g) significantly higher than those of the individual solute solutions. Mixing of the amino acid with some dicarboxylic acids (e.g., oxalic acid) also suggested an upward shift of the transition temperature. Contrarily, combinations of the amino acid with monocarboxylic acids (e.g., acetic acid) had T'gs between those of the individual solute solutions. Co-lyophilization of the basic amino acids and citric acid or L-tartaric acid resulted in amorphous solids that have glass transition temperatures (Tg) higher than the individual components. Mid- and near-infrared analysis indicated altered environment around the functional groups of the consisting molecules. Some of the glass-state excipient combinations protected an enzyme (lactate dehydrogenase, LDH) from inactivation during freeze-drying. The glass-state excipient combinations formed by hydrogen-bonding and electrostatic interaction network would be potent alternative to stabilize therapeutic proteins in freeze-dried formulations.

Molecular Dynamics Simulations of Rhodopsin Point Mutants at the Cytoplasmic Side of Helices 3 and 6

The present work reports on a structural analysis carried out through different computer simulations of a set of rhodopsin mutants with differential functional features in regard to the wild type. Most of these mutants, whose experimental features had previously been reported [Ramon et al. J Biol Chem 282, 14272–14282 (2007)], were designed to perturb a network of electrostatic interactions located at the cytoplasmic sides of transmembrane helices 3 and 6. Geometric and energetic features derived from the detailed analysis of a series of molecular dynamics simulations of the different rhodopsin mutants, involving positions 134(3.49), 247(6.30), and 251(6.34), suggest that the protein structure is sensitive to these mutations through the local changes induced that extend further to the secondary structure of neighboring helices and, ultimately, to the packing of the helical bundle. Overall, the results obtained highlight the complexity of the analyzed network of electrostatic interactions where the effect of each mutation on protein structure can produce rather specific features.

Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp(203)/Trp(205)/Trp(206)) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan 'anchor' was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

Crystal Structure and Raman Studies of dsFP483, a Cyan Fluorescent Protein from Discosoma striata

To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins (FPs), we determined the 2.1-Å X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan class of Anthozoa FPs and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437–440 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the protein-intrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stability. The refined crystal structure of the biological tetramer (space group C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical with that in green fluorescent protein (GFP). To test the roles of specific residues in color modulation, we investigated the optical properties of the H163Q and K70M variants. Although absorbance bands remain broad, peak excitation maxima are red shifted to 455 and 460 nm, emitting cyan light and green light, respectively. To probe chromophore ground-state features, we collected Raman spectra using 752-nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral GFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited-state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.

Probing electrostatic interactions and ligand binding in aspartyl‐tRNA synthetase through site‐directed mutagenesis and computer simulations

Faithful genetic code translation requires that each aminoacyl-tRNA synthetase recognise its cognate amino acid ligand specifically. Aspartyl-tRNA synthetase (AspRS) distinguishes between its negatively-charged Asp substrate and two competitors, neutral Asn and di-negative succinate, using a complex network of electrostatic interactions. Here, we used molecular dynamics simulations and site-directed mutagenesis experiments to probe these interactions further. We attempt to decrease the Asp/Asn binding free energy difference via single, double and triple mutations that reduce the net positive charge in the active site of Escherichia coli AspRS. Earlier, Glutamine 199 was changed to a negatively-charged glutamate, giving a computed reduction in Asp affinity in good agreement with experiment. Here, Lysine 198 was changed to a neutral leucine; then, Lys198 and Gln199 were mutated simultaneously. Both mutants are predicted to have reduced Asp binding and improved Asn binding, but the changes are insufficient to overcome the initial, high specificity of the native enzyme, which retains a preference for Asp. Probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments, we found no detectable activity for the mutant enzymes, indicating weaker Asp binding and/or poorer transition state stabilization. The simulations show that the mutations' effect is partly offset by proton uptake by a nearby histidine. Therefore, we performed additional simulations where the nearby Histidines 448 and 449 were mutated to neutral or negative residues: (Lys198Leu, His448Gln, His449Gln), and (Lys198Leu, His448Glu, His449Gln). This led to unexpected conformational changes and loss of active site preorganization, suggesting that the AspRS active site has a limited structural tolerance for electrostatic modifications. The data give insights into the complex electrostatic network in the AspRS active site and illustrate the difficulty in engineering charged-to-neutral changes of the preferred ligand.

The relative energetic contributions of dominant P1 pocket versus hydrogen bonding interactions to peptide:class II stability: Implications for the mechanism of DM function

Peptides are bound to MHC class II molecules by an array of hydrogen bonds between conserved MHC class II protein side-chains and the peptide backbone and through interactions between MHC protein pockets and peptide side-chain anchors. The crystal structure of murine I-A k protein with peptide shows a network of electrostatic interactions with the P1 aspartic acid anchor and an arginine in the P1 pocket that are thought to constitute the major stabilizing interaction between peptide and MHC. In this paper, have explored the relative energetic contribution of this dominant P1 pocket interaction with that made by a genetically conserved hydrogen bond which is formed by the beta 81 histidine residue and the main chain of the bound peptide. We have performed peptide dissociation experiments using antigenic peptides or variants that have altered side-chain interactions with the I-A k P1 pocket using either native I-A k or I-A k proteins mutated to disrupt the N-terminal hydrogen bond. The results demonstrate that the N-terminal hydrogen bonds in I-A k complexes make highly significant energetic contributions to the kinetic stabilities comparable to or greater than the energetic contribution of highly favorable P1 pocket interactions. Hence, we conclude that the kinetic stability of MHC class II:peptide complexes critically depends on two quite distinct molecular interactions between peptide and MHC located at the peptide's amino terminus. We discuss these results in light of the proposed mechanism for DM function.

Non‐charged amino acids from three different domains contribute to link agonist binding to channel gating in α7 nicotinic acetylcholine receptors

Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.

Critical Role of Electrostatic Interactions of Amino Acids at the Cytoplasmic Region of Helices 3 and 6 in Rhodopsin Conformational Properties and Activation

The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.

Molecular basis of coiled-coil formation.

Coiled coils have attracted considerable interest as design templates in a wide range of applications. Successful coiled-coil design strategies therefore require a detailed understanding of coiled-coil folding. One common feature shared by coiled coils is the presence of a short autonomous helical folding unit, termed "trigger sequence," that is indispensable for folding. Detailed knowledge of trigger sequences at the molecular level is thus key to a general understanding of coiled-coil formation. Using a multidisciplinary approach, we identify and characterize here the molecular determinants that specify the helical conformation of the monomeric early folding intermediate of the GCN4 coiled coil. We demonstrate that a network of hydrogen-bonding and electrostatic interactions stabilize the trigger-sequence helix. This network is rearranged in the final dimeric coiled-coil structure, and its destabilization significantly slows down GCN4 leucine zipper folding. Our findings provide a general explanation for the molecular mechanism of coiled-coil formation.

Mechanism of peptide bond formation on the ribosome

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.

Molecular Dynamics Simulations Show That Bound Mg2+ Contributes to Amino Acid and Aminoacyl Adenylate Binding Specificity in Aspartyl-tRNA Synthetase through Long Range Electrostatic Interactions

Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg2+ cations in an unusual, bent geometry. The Mg2+ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP x Mg3(2+) complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg2+ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg2+ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg2+ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.

Docking Interactions Induce Exposure of Activation Loop in the MAP Kinase ERK2

MAP kinases bind activating kinases, phosphatases, and substrates through docking interactions. Here, we report a 1.9 A crystallographic analysis of inactive ERK2 bound to a "D motif" docking peptide (pepHePTP) derived from hematopoietic tyrosine phosphatase, a negative regulator of ERK2. In this complex, the complete D motif interaction defined by mutagenic analysis is observed, including extensive electrostatic interactions with the "CD" site of the kinase. Large conformational changes occur in the activation loop where the dual phosphorylation sites, which are buried in the inactive form of ERK2, become exposed to solvent in the complex. Similar conformational changes occur in a complex between ERK2 and a MEK2 (MAP/ERK kinase-2)-derived D motif peptide (pepMEK2). D motif peptides are known to bind homologous loci in the MAP kinases p38alpha and JNK1, also inducing conformational changes in these enzymes. However, the binding interactions and conformational changes are unique to each, thus contributing to specificity among MAP kinases.

Platinum Complexes with the Novel Ligand Diethyl [(Methylsulfinyl)‐methyl]phosphonate (SMP): Solid‐State Characterization of Potassium Trichloro(SMP)platinum(II) which, in Solution, Gives Dichloro(SMP)‐platinum(II) and Potassium Chloride

AbstractPhosphonate ligands have been employed in the synthesis of platinum complexes, which are active against bone tumors. The stability of a new compound of this family, [PtCl2(SMP)] {1; SMP = diethyl[(methylsulfinyl)methyl]phosphonate}, has been investigated in water and acetone. The compound is stable in aqueous solution where it undergoes only partial solvolysis that is completely repressed by addition of free chloride ions. However, crystallization of 1 from water/acetone/chloroform (0.2:1:1, v/v/v) containing an equimolar amount of KCl affords a new compound containing monodentate SMP {K[PtCl3(SMP‐S)], 2}. Dissolution of 2 in water or acetone restores 1. The driving force in the formation of 2 appears to be the network of electrostatic interactions between cations and complex anions in the solid state. It is not only the detached P=O oxygen atom, but also the oxygen atom bound to the sulfur atom and a coordinated chloride ligand of the same platinum unit that interact rather strongly with the cation. It is expected that the same reaction (partial detachment of the phosphonate and anchoring of the cation) can take place in hypercalcaemic districts associated to bone tumors. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Hierarchical self-assembly of water-soluble porphyrins

Hierarchy is a powerful and promising tool to rationally synthesize non-covalent complex species. In this paper we show two different examples of hierarchically driven aggregation processes allowing for the control of aggregation state or “shape” of the final species. In the first case we propose that water-soluble porphyrins presenting various protonation steps can follow kinetic routes alternative to the thermodynamic pathway; namely for each protonation step is allowed, in principle, an alternative kinetically driven homo-self-aggregation process. Our results indicate that the thermodynamic route leads to a monomeric, protonated final state, but the kinetic pathway ends with self-assemblies of the title porphyrin. The bias between the two final states can be easily pre-determined by going quickly or slowly throughout the thermodynamic–kinetic junction.The second case deals with the so-called “chiral memory” phenomenon. Also in this case the role of the kinetic control over the assembly process is remarkable. In particular, we have observed that self-aggregation of opposite charged, achiral porphyrins does not lead to induced chirality even if a chiral template is added after their aggregation. The final aggregates are kinetically inert, mostly because stabilized by a network of electrostatic interactions between net charges. However, if the two supramolecular components are mixed in the presence of a chiral template, then an induced circular dichroism signal (ICD) appears in the Soret region (the main absorption feature of porphyrins in the visible region whose maximum and intensity are strongly affected by the aggregation state). Removal of the template does not significantly affect the intensity of the ICD: the inert aggregates have memorized the chiral template shape. Remarkably, these species – now inherently chiral – act as very efficient templates for self-aggregation of additional non-chiral porphyrins.

Stereochemical and electronic interaction studies of some meta- and para-substituted α-methylsulfinyl-α-diethoxyphosphorylacetophenones

The IR and 1H NMR spectra analyses of some meta- (Y=OMe 1, Me 2, F 3, Cl 4, Br 5 and NO 2 6) and para- (Y=OMe 7, H 8, F 9, Cl 10, Br 11 and NO 2 12) substituted α-methylsulfinyl-α-diethoxyphosphorylacetophenones m,p-YPhC(O)C*H[S*(O)Me][P(O)(OEt) 2], along with theoretical calculations at the B3LYP/6-31+g(d,p) level, for compound 8, have shown, in solution of solvents of increasing polarity and in the gas phase, the existence of two enantiomeric pairs (racemic mixtures). The more stable and less polar (C S S S ; C R S R ) enantiomeric pair (conformer pair I) display the [MeS(O)] group in a quasi-periplanar ( quasi-cis) geometry and the [P(O)(OEt) 2] group in a anti-clinal ( gauche) geometry relative to the carbonyl group. The less stable and more polar (C R S S ; C S S R ) enantiomeric pair (conformer pair II) presents both substituents in a syn-clinal ( gauche) geometry. The observed solvent effect on the IR ν CO doublet components and on the two 1H NMR methyl signals relative intensities, for the whole series along with the complete deuterium exchange of the α-methine hydrogen atom of 1, strongly indicate that both pairs of diastereomers [(C S S S ; C R S R ) and (C R S S ; C S S R )] are equilibrated in solution through an intermediate enolic form. The more stable (C S S S ; C R S R ) enantiomeric pair [conformer pair ( I)] is strongly stabilised by O (CO) δ−⋯S (SO) δ+ charge transfer and electrostatic interaction, while the remaining electronic interactions are almost counterbalanced among themselves, and are important in the stabilisation of the less stable (C R S S ; C S S R ) enantiomeric pair [conformer pair ( II)]. Compound 1, in the solid state, occurs as the (C R S S ; C S S R ) enantiomeric pair [conformer pair ( II′)], as determined by X-ray diffraction analysis, whose geometry is similar to conformer pair ( II), found in gas phase. The O (CO) δ−⋯S (SO) δ+, O (POEt) δ−⋯C (CO) δ+, O (POEt) δ−⋯S (SO) δ+ and H (o-Ph) δ+⋯O (CO) δ− intramolecular interactions contribute for the stabilisation of conformer pair ( II′) in the solid. Further stabilisation derives from dipole moment couplings and by an intricate network of electrostatic interactions (hydrogen bonds).

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface.

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.

Electrostatic interactions across the dimer–dimer interface contribute to the pH-dependent stability of a tetrameric malate dehydrogenase

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic bacteria usually are dimers. Using site-directed mutagenesis, we show here that a network of electrostatic interactions across the extra dimer–dimer interface in CaMDH is important for thermal stability and oligomeric integrity. Stability effects of single point mutations (E25Q, E25K, D56N, D56K) varied from −1.2°C to −26.8°C, and depended strongly on pH. Gel-filtration experiments indicated that the 26.8°C loss in stability observed for the D56K mutant at low pH was accompanied by a shift towards a lower oligomerization state.

GroEL Stability and Function: CONTRIBUTION OF THE IONIC INTERACTIONS AT THE INTER-RING CONTACT SITES

The chaperonin GroEL consists of a double ring structure made of identical subunits that display different modes of allosteric communication. The protein folding cycle requires the simultaneous positive intra-ring and negative inter-ring cooperativities of ATP binding. This ensures GroES binding to one ring and release of the ligands from the opposite one. To better characterize inter-ring allosterism, the thermal stability as well as the temperature dependence of the functional and conformational properties of wild type GroEL, a single ring mutant (SR1) and two single point mutants suppressing one interring salt bridge (E434K and E461K) were studied. The results indicate that ionic interactions at the two interring contact sites are essential to maintain the negative cooperativity for protein substrate binding and to set the protein thermostat at 39 degrees C. These electrostatic interactions contribute distinctly to the stability of the inter-ring interface and the overall protein stability, e.g. the E434K thermal inactivation curve is shifted to lower temperatures, and its unfolding temperature and activation energy are also lowered. An analysis of the ionic interactions at the inter-ring contact sites reveals that at the so called "left site" a network of electrostatic interactions involving three charged residues might be established, in contrast to what is found at the "right site" where only two oppositely charged residues interact. Our data suggest that electrostatic interactions stabilize protein-protein interfaces depending on both the number of ionic interactions and the number of residues engaged in each of these interactions. In the case of GroEL, this combination sets the thermostat of the protein so that the chaperonin distinguishes physiological from stress temperatures.

Deciphering the Role of Individual Acyl Chains in the Interaction Network between Phosphatidylserines and a Single-Spanning Membrane Protein

PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts.