Hydrophobic and electrostatic interactions modulate protein escape at the ribosomal exit tunnel

After translation, nascent proteins must escape the ribosomal exit tunnel to attain complete folding to their native states. This escape process also frees up the ribosome tunnel for a new translation job. In this study, we investigate the impacts of energetic interactions between the ribosomal exit tunnel and nascent proteins on the protein escape process by molecular dynamics simulations using partially coarse-grained models that incorporate hydrophobic and electrostatic interactions of the ribosome tunnel of Haloarcula marismortui with nascent proteins. We find that, in general, attractive interactions slow down the protein escape process, whereas repulsive interactions speed it up. For the small globular proteins considered, the median escape time correlates with both the number of hydrophobic residues, N_h, and the net charge, Q, of a nascent protein. A correlation coefficient exceeding 0.96 is found for the relation between the median escape time and a combined quantity of N_h + 5.9Q, suggesting that it is ∼6 times more efficient to modulate the escape time by changing the total charge than the number of hydrophobic residues. The estimated median escape times are found in the submillisecond-to-millisecond range, indicating that the escape does not delay the ribosome recycling. For various types of the tunnel model, with and without hydrophobic and electrostatic interactions, the escape time distribution always follows a simple diffusion model that describes the escape process as a downhill drift of a Brownian particle, suggesting that nascent proteins escape along barrier-less pathways at the ribosome tunnel.     

Acidic groups docked to well defined wetted pockets at the core of the binding interface: a tale of scoring and missing protein interactions in CAPRI

Some challenging targets in CAPRI (T24/25 and T26) involve binding solvent accessible acidic residues at the core of the binding interface, where they are always found immersed in crystal waters. In fact, Asp and Glu residues are more likely to form part of the hydrogen bond network of their surrounding crystal water molecules than to form a buried salt bridge. Interestingly, many of the crystal waters mediating the intermolecular interactions of the acidic groups are already present in the unbound structure, reinforcing the notion that some water molecules behave as an extension of the protein structure. This is in contrast to acidic groups found in the periphery of the binding interface that form ubiquitous salt bridges that cement the high affinity complex, while at the same time they are exposed to rapidly exchanging water molecules. Because of this, dichotomy implicit solvent scoring functions fail to properly rank these complexes by prioritizing salt bridges rather than water mediated contacts. A detailed analysis of Target 24, for which our group predicted two out of the four successful homology model complex structures, and Target 26 reveal how crystal waters shape the binding cavities of acidic groups prior to binding, in agreement with the theory of anchor residues as mediators of protein recognition.     

Electrostatic interactions in an integral membrane protein

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.     

Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein

Despite the fact that the porcine odorant-binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 A) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (lambda(max) = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (lambda(max) = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat-induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I(320)/I(365)) upon excitation at lambda(ex) = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85 degrees C, and the calculated midpoint of transition was centered at 70 degrees C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature-induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature.     

Hydrophobic helical hairpins: design and packing interactions in membrane environments

Helix-helix interactions within membranes are dominated by van der Waals packing motifs and side chain-side chain hydrogen bond formation, which act in tandem to determine the residues that comprise the interface between two given helices. To explore in a systematic manner the tertiary contacts between transmembrane helices, we have designed and expressed in Escherichia coli highly hydrophobic helix-loop-helix constructs of prototypic sequence K(1)KKKKKKFAIAIAIIAWAX(19)AIIAIAIAIKSPGSKIAIAIAIIAZ(44)AWAIIAIAIAFKKKKKKK(62), where "small" (Ala) and "large" (Ile) residues were used to maximize the tertiary contact area. Evidence that the two transmembrane (TM) segments in the AI construct contain an interface conducive for folding into a hairpin structure was obtained from the results that (i) the single TM AI(pep) peptide derived from the AI hairpin forms SDS-resistant dimers on PAGE gels and (ii) the corresponding sequence forms a strong dimer when examined in vivo in TOXCAT assays. Site-directed mutagenesis of AI hairpins was carried out to incorporate each of the 20 commonly occurring amino acids at X positions. Analysis on Western blots using an oligomerization assay in 12% NuPage-sodium dodecyl sulfate (SDS) indicated that mutants with X = E, D, Q, R, N, H, and K largely formed SDS-resistant dimers-which likely correspond to H-bonded four-helix bundles-while all the others (e.g., X = F, W, L, I, M, V, C, Y, A, T, S, G, and P) remained monomeric. Systematic studies of X/Z double mutants indicated that formation of hairpin dimers is the result of the disruption of stabilizing interactions between the antiparallel helices within the AI construct. The overall results suggest that, in situations where hydrophobic van der Waals packing energy between helices is sufficient to prevent significant rotation about the major axes of interacting helices, intrahairpin side chain-side chain H-bond formation will occur mainly when pairs of polar residues are interfacially located and proximal. Knowledge of the relative contributions of these forces should be of value, for example, in clarifying the context--and the structural consequences--of disease-related mutations.     

Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains

Synaptotagmin I is a synaptic vesicle associated membrane protein that appears to regulate Ca(2+)-mediated exocytosis. Here, the Ca(2+)-dependent membrane interactions of a water soluble fragment of synaptotagmin I (C2AB) that contains its two C2 domains (C2A and C2B) were determined using site-directed spin labeling. Membrane depth parameters were obtained for 19 spin-labeled mutants of C2AB when bound to phosphatidylcholine and phosphatidylserine membranes, and these distance constraints were used in combination with the high-resolution structures of C2A and C2B to generate a model for the membrane orientation and position of synaptotagmin at the bilayer interface. Both C2A and C2B bind to the membrane interface with their first and third Ca(2+) binding loops penetrating the membrane interface. The polybasic face of C2B does not interact with the membrane lipid but is available for electrostatic interaction with other components of the fusion machinery. When compared to positions determined previously for the isolated domains, both C2A and C2B have similar orientations; however, the two domains are positioned deeper into the bilayer interior when present in the tandem construct. These data indicate that C2A and C2B do not act independently but influence their mutual membrane penetration. This may explain the occurrence of multiple C2 domains in proteins that function in membrane trafficking and repair.     

Enzyme-inhibitor interactions at the plant-pathogen interface

The plant apoplast during plant-pathogen interactions is an ancient battleground that holds an intriguing range of attacking enzymes and counteracting inhibitors. Examples are pathogen xylanases and polygalacturonases that are inhibited by plant proteins like TAXI, XIP, and PGIP; and plant glucanases and proteases, which are targeted by pathogen proteins such as GIP1, EPI1, EPIC2B, and AVR2. These seven well-characterized inhibitors have different modes of action and many probably evolved from inactive enzymes themselves. Detailed studies of the structures, sequence variation, and mutated proteins uncovered molecular struggles between these enzymes and their inhibitors, resulting in positive selection for variant residues at the contact surface, where single residues determine the outcome of the interaction.     

Plant-insect dialogs: complex interactions at the plant-insect interface

Although five different classes of insect herbivore-produced elicitors of plant volatiles have been identified, this is only a part of the complex, chemically mediated interactions between insect herbivores and their host plants. The defensive reactions of the plant, following physical injury by the herbivore, are influenced by a multitude of factors including, but not necessarily limited to, the elicitors and numerous other herbivore-associated molecules, as well as microbes on the plant surface that may alter plant defensive pathways. Ultimately, a thorough and accurate understanding of the chemical ecology of insect-plant interactions will require a more holistic approach, taking into consideration the ecological and physiological context in which a plant perceives and responds to herbivore-associated signals.     

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1 , a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the γ-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21 , we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20 .     

Asymmetric interactions in the adenosine-binding pockets of the MS2 coat protein dimer

Background  

The X-ray structure of the MS2 coat protein-operator RNA complex reveals the existence of quasi-synmetric interactions of adenosines -4 and -10 in pockets formed on different subunits of the coat protein dimer. Both pockets utilize the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59, and Lys61. We call these sites the adenosine-binding pockets.     

Lipids at the interface of virus-host interactions

Viruses physically and metabolically remodel the host cell to establish an optimal environment for their replication. Many of these processes involve the manipulation of lipid signaling, synthesis, and metabolism. An emerging theme is that these lipid-modifying pathways are also linked to innate antiviral responses and can be modulated to inhibit viral replication.     

The importance of the membrane interface as the reference state for membrane protein stability

The insertion of nascent polypeptide chains into lipid bilayer membranes and the stability of membrane proteins crucially depend on the equilibrium partitioning of polypeptides. For this, the transfer of full sequences of amino-acid residues into the bilayer, rather than individual amino acids, must be understood. Earlier studies have revealed that the most likely reference state for partitioning very hydrophobic sequences is the membrane interface. We have used μs-scale simulations to calculate the interface-to-transmembrane partitioning free energies ΔG_S→TM for two hydrophobic carrier sequences in order to estimate the insertion free energy for all 20 amino acid residues when bonded to the center of a partitioning hydrophobic peptide. Our results show that prior single-residue scales likely overestimate the partitioning free energies of polypeptides. The correlation of ΔG_S→TM with experimental full-peptide translocon insertion data is high, suggesting an important role for the membrane interface in translocon-based insertion. The choice of carrier sequence greatly modulates the contribution of each single-residue mutation to the overall partitioning free energy. Our results demonstrate the importance of quantifying the observed full-peptide partitioning equilibrium, which is between membrane interface and transmembrane inserted, rather than combining individual water-to-membrane amino acid transfer free energies.     

DNA protein interactions at the interferon-responsive promoter elements: potential for an H-DNA conformation.

The regions of several genes (IFI-56K, HLA-A3, HLA-DR and 6-16) containing the (putative) ISRE (Interferon Stimulatable Response Element) were tested for their ability to be recognized by HeLa cells nuclear extract proteins. In a band shift assay, all probes yielded two B1 and B2 DNA-protein complexes of similar mobilities. Unexpectedly the titration of the B1 complex with a synthetic ISRE core (OL1), promoted the formation of B2. Both the probe and OL1 were recovered in B2. For each probe, the possibility of the part of the sequence involved in B1 complex to form a H-DNA structure with the part of the sequence involved in B2 exists. Such a structure was favored by the colinearity of the pairing regions and requires ATP. Although probes seemed to have a secondary structure, the formal existence of a H-DNA structure has not been demonstrated. Such a model could be extended to other interferon inducible gene promoters and may account for their binding properties and differential inducibility after 5' deletion or point mutations.     

Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atomic tritium as an instrument for study of protein behavior at the air-water interface

Atomic tritium was successfully applied as an instrument for study of protein behavior at the air-water interface. Samples of lysozyme solution in 20 mM phosphate buffer (pH 7.0) with concentration of 2 mg/ml incubated at the room temperature for 1 h were exposed to bombardment with tritium atoms generated on hot tungsten wire in special vacuum device. This procedure resulted in substitution of hydrogen atoms by radioactive tritium in the thin surface layer of studied preparations. Analysis of experimental data on intramolecular radioactivity distribution in lysozyme and computer simulation of tritium bombardment allowed us to suggest two equally probable opposite orientations of lysozyme molecule in the adsorption layer at the air-water interface.     

Low dielectric permittivity of water at the membrane interface: effect on the energy coupling mechanism in biological membranes

Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (deltapH(B)) or between two membrane surfaces (deltapH(S)). To scrutinize whether deltapH(S) can deviate from deltapH(B), we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, PHYS: Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5-1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2-0.3 eV) than for monovalent cations (0.1-0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH(S) of approximately 6.0; the value of pH(S) was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.