Small, monomeric G proteins in plants

The superfamily of small, monomeric GTP-binding proteins, in Arabidopsis thaliana comprising 93 members, is classified into four families: Arf/Sar, Rab, Rop/Rac, and Ran families. All monomeric G proteins function as molecular switches that are activated by GTP and inactivated by the hydrolysis of GTP to GDP. GTP/GDP cycling is controlled by three classes of regulatory protein: guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). Proteins of Arf family are primarily involved in regulation of membrane traffic and organization of the cytoskeleton. Arf1/Sar1 proteins regulate the formation of vesicle coat at different steps in the exocytic and endocytic pathways. Rab GTPases are regulators of vesicular transport. They are involved in vesicle formation, recruitment of cytoskeletal motor proteins, and in vesicle tethering and fusion. Rop proteins serve as key regulators of cytoskeletal reorganization in response to extracellular signals. Several data have also shown that Rop proteins play additional roles in membrane trafficking and regulation of enzymes activity. Ran proteins are involved in nucleocytoplasmic transport.     

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.     

Thermal stability of proteins in intermolecular complexes.

A general phenomenological model is proposed for the estimation of the influence of the formation of complexes with ligands on thermal stability of proteins. In this model the reversible processes of unfolding-refolding and of association-dissociation of protein-ligand complexes and of the irreversible chemical degradation of the unfolded protein were analyzed jointly. By using certain approximations, the analytical expressions for both the thermodynamic and kinetic stabilization are obtained. Two thermodynamic and four kinetic regimes of stabilization and destabilization can exist in such system. Each thermodynamic regime appears to be compatible with three different kinetic regimes. The effect of the formation of complexes on thermodynamic and kinetic stability of the protein is determined by the degrees of binding of the ligand to the folded and unfolded protein species and by the rates of irreversible degradation of free protein and protein in complex.     

Discriminating between homodimeric and monomeric proteins in the crystalline state

Scores calculated from intermolecular contacts of proteins in the crystalline state are used to differentiate monomeric and homodimeric proteins, by classification into two categories separated by a cut-off score value. The generalized classification error is estimated by using bootstrap re-sampling on a nonredundant set of 172 water-soluble proteins whose prevalent quaternary state in solution is known to be either monomeric or homodimeric. A statistical potential, based on atom-pair frequencies across interfaces observed with homodimers, is found to yield an error rate of 12.5%. This indicates a small but significant improvement over the measure of solvent accessible surface area buried in the contact interface, which achieves an error rate of 15.4%. A further modification of the latter parameter relating the two most extensive contacts of the crystal results in an even lower error rate of 11.1%.     

Alpha-helix stability in proteins. II. Factors that influence stability at an internal position.

The solvent-exposed residue Ala32 in the second alpha-helix of barnase was replaced by all other naturally occurring amino acids and the concomitant effects on the protein stability were determined. The results are assumed to reflect both the distinct conformational preferences of the different amino acids and also possible intrahelical interactions. The conformational preferences may be fully rationalized by invoking only a few physical principles. The results agree well with recently experimentally determined rank-order of helix-forming tendencies determined on a model peptide. There is very weak correlation between the results and the experimental host-guest values. There is a weak correlation between our results and the statistical helix propensities and a slightly better correlation with the positional-dependent statistical parameters of J. S. Richardson, and D. C. Richardson.     

Conformational stability of adrenodoxin mutant proteins.

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.     

The fluorescent monomeric protein Kusabira Orange. Pressure effect on its structure and stability

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network.     

Salt stress proteins induced in Listeria monocytogenes

The ability of Listeria monocytogenes to tolerate salt stress is of particular importance, as this pathogen is often exposed to such environments during both food processing and food preservation. In order to understand the survival mechanisms of L. monocytogenes, an initial approach using two-dimensional polyacrylamide gel electrophoresis was performed to analyze the pattern of protein synthesis in response to salt stress. Of 400 to 500 visible proteins, the synthesis of 40 proteins (P < 0.05) was repressed or induced at a higher rate during salt stress. Some of the proteins were identified on the basis of mass spectrometry or N-terminal sequence analysis and database searching. Twelve proteins showing high induction after salt stress were similar to general stress proteins (Ctc and DnaK), transporters (GbuA and mannose-specific phosphotransferase system enzyme IIAB), and general metabolism proteins (alanine dehydrogenase, CcpA, CysK, EF-Tu, Gap, GuaB, PdhA, and PdhD).     

Salt bridge interactions: Stability of the ionic and neutral complexes in the gas phase,in solution,and in proteins

A theoretical study on the stability of the salt bridges in the gas phase, in solution, and in the interior of proteins is presented. The study is mainly focused on the interaction between acetate and methylguanidinium ions, which were used as model compounds for the salt bridge between Asp (Glu) and Arg. Two different solvents (water and chloroform) were used to analyze the effect of varying the dielectric constant of the surrounding media on the salt bridge interaction. Calculations in protein environments were performed by using a set of selected protein crystal structures. In all cases attention was paid to the difference in stability between the ion pair and neutral hydrogen-bonded forms. Comparison of the results determined in the gas phase and in solution allows us to stress the large influence of the environment on the binding process, as well as on the relative stability between the ionic and neutral complexes. The high anisotropy of proteins and the local microenvironment in the interior of proteins make a decisive contribution in modulating the energetics of the salt bridge. In general, the formation of salt bridges in proteins is not particularly favored, with the ion pair structure being preferred over the interaction between neutral species. Proteins 32:67–79, 1998. © 1998 Wiley-Liss, Inc.     

Interactions between bacterial flagellar axial proteins in their monomeric state in solution

The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found interactions in the following two cases only. Flagellin facilitated HAP3 aggregation into beta-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2-HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the heterodimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum.     

Mammalian facilitative glucose transporters: evidence for similar substrate recognition sites in functionally monomeric proteins.

Four facilitative glucose transporters isoforms, GLUT1/erythrocyte, GLUT2/liver, GLUT3/brain, and GLUT4/muscle-fat, as well as chimeric transporter proteins were expressed in Xenopus oocytes, and their properties were studied. The relative Km's of the transporters for 2-deoxyglucose were GLUT3 (Km = 1.8 mM) > GLUT4 (Km = 4.6 mM) > GLUT1 (Km = 6.9 mM) > GLUT2 (Km = 17.1 mM). In a similar fashion, the uptake of 2-deoxyglucose by GLUT1-, GLUT2-, and GLUT3-expressing oocytes was inhibited by a series of unlabeled hexoses and pentoses and by cytochalasin B in a similar hierarchical order. To determine if the functional unit of the glucose transporter was a monomer or higher-order multimer, the high-affinity transporter GLUT3 was coexpressed with either the low-affinity GLUT2 or a GLUT3 mutant which contained a transport inactivating Trp410-->Leu substitution. In oocytes expressing both GLUT2 and GLUT3, the transport activity associated with each transporter isoform could be distinguished kinetically. Similarly, there was no alteration in the kinetic parameters of GLUT3, or the ability of glucose or cytochalasin B to inhibit 2-deoxyglucose uptake, when coexpressed with up to a 3-fold greater amount of functionally inactive mutant of GLUT3. These studies suggest that the family of glucose transporters have similar binding sites which may be in the form of a functional monomeric unit when expressed in Xenopus oocytes.     

Superfamily of monomeric GTP-binding proteins in plants: 1. Role of rop proteins in the control of plant growth and development

The control of plant growth, differentiation, and development is considered in relation to the involvement of monomeric GTP-binding proteins (mG-proteins) in the extra-and intracellular signal transduction. The principal attention is paid to Rop mG-proteins, unique small GTPases of eukaryotic cells functioning during various developmental stages of plants, from pollen tube and root hair growth to plant responses to biotic and abiotic stresses.     

Exchange and stability of HeLa ribosomal proteins in vivo.

The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.     

Salt dependence of nucleic acid hairpin stability

Single-stranded junctions/loops are frequently occurring structural motifs in nucleic acid structures. Due to the polyanionic nature of the nucleic acid backbone, metal ions play a crucial role in the loop stability. Here we use the tightly bound ion theory, which can account for the possible ion correlation and ensemble (fluctuation) effects, to predict the ion-dependence of loop and stem-loop (hairpin) free energies. The predicted loop free energy is a function of the loop length, the loop end-to-end distance, and the ion (Na⁺ and Mg²⁺ in this study) concentrations. Based on the statistical mechanical calculations, we derive a set of empirical formulas for the loop thermodynamic parameters as functions of Na⁺ and Mg²⁺ concentrations. For three specific types of loops, namely, hairpin, bulge, and internal loops, the predicted free energies agree with the experimental data. Further applications of these empirical formulas to RNA and DNA hairpin stability lead to good agreements with the available experimental data. Our results indicate that the ion-dependent loop stability makes significant contribution to the overall ion-dependence of the hairpin stability.     

Quenching in Arabidopsis thaliana mutants lacking monomeric antenna proteins of photosystem II

The minor light-harvesting complexes CP24, CP26, and CP29 have been proposed to play a key role in the zeaxanthin (Zx)-dependent high light-induced regulation (NPQ) of excitation energy in higher plants. To characterize the detailed roles of these minor complexes in NPQ and to determine their specific quenching effects we have studied the ultrafast fluorescence kinetics in knockout (ko) mutants koCP26, koCP29, and the double mutant koCP24/CP26. The data provide detailed insight into the quenching processes and the reorganization of the Photosystem (PS) II supercomplex under quenching conditions. All genotypes showed two NPQ quenching sites. Quenching site Q1 is formed by a light-induced functional detachment of parts of the PSII supercomplex and a pronounced quenching of the detached antenna parts. The antenna remaining bound to the PSII core was also quenched substantially in all genotypes under NPQ conditions (quenching site Q2) as compared with the dark-adapted state. The latter quenching was about equally strong in koCP26 and the koCP24/CP26 mutants as in the WT. Q2 quenching was substantially reduced, however, in koCP29 mutants suggesting a key role for CP29 in the total NPQ. The observed quenching effects in the knockout mutants are complicated by the fact that other minor antenna complexes do compensate in part for the lack of the CP24 and/or CP29 complexes. Their lack also causes some LHCII dissociation already in the dark.     

Rational design of true monomeric and bright photoactivatable fluorescent proteins

Monomeric (m)Eos2 is an engineered photoactivatable fluorescent protein widely used for super-resolution microscopy. We show that mEos2 forms oligomers at high concentrations and forms aggregates when labeling membrane proteins, limiting its application as a fusion partner. We solved the crystal structure of tetrameric mEos2 and rationally designed improved versions, mEos3.1 and mEos3.2, that are truly monomeric, are brighter, mature faster and exhibit higher photon budget and label density.     

A novel type of allosteric regulation: functional cooperativity in monomeric proteins

Cooperative functional properties and allosteric regulation in cytochromes P450 play an important role in xenobiotic metabolism and define one of the main mechanisms of drug-drug interactions. Recent experimental results suggest that ability to bind simultaneously two or more small organic molecules can be the essential feature of cytochrome P450 fold, and often results in rich and complex pattern of allosteric behavior. Manifestations of non-Michaelis kinetics include homotropic and heterotropic activation and inhibition effects depending on the stoichiometric ratios of substrate and effector, changes in the regio- and stereospecificity of catalytic transformations, and often give rise to the clinically important drug-drug interactions. In addition, functional response of P450 systems is modulated by the presence of specific and non-specific effector molecules, metal ions, membrane incorporation, formation of homo- and hetero-oligomers, and interactions with the protein redox partners. In this article we briefly overview the main factors contributing to the allosteric effects in cytochromes P450 with the main focus on the sources of cooperative behavior in xenobiotic metabolizing monomeric heme enzymes with their conformational flexibility and extremely broad substrate specificity. The novel mechanism of functional cooperativity in P450 enzymes does not require substantial binding cooperativity, rather it implies the presence of one or more binding sites with higher affinity than the single catalytically active site in the vicinity of the heme iron.