Biochemical characterization of different conformational states of the Sf9 cell-purified p53His175 mutant protein

In this study, we expressed and purified the p53 mutant encoded by the His175 allele (p53His175) in a baculovirus expression system in order to study the folding and the DNA binding activity of the protein. A two-site ELISA revealed that purified p53His175 protein preferentially displayed a PAb1620 conformation, which appeared to be not sufficient to interact specifically with DNA. The cryptic DNA binding activity of this mutant was then investigated by electrophoretic mobility shift assay in the presence of anti-p53 antibodies, and shown to be refractory to significant activation by PAb421 (a potent allosteric activator of wild-type p53's DNA binding activity). Nevertheless, p53His175 DNA binding was regulated by antibodies targeting the N-terminal region of the protein. Furthermore, while the protein preferentially displayed a PAb1620 conformation, our data suggested the existence of an equilibrium between at least two folding states of the protein (PAb1620 and PAb240 conformations). A model rationalizing the conformation, antibody-interacting ability and DNA binding regulation potential of p53His175 is presented.     

The mitochondrial oxoglutarate carrier: structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling

The structural and dynamic features of the fourth transmembrane segment of the mitochondrial oxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance (EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutive residues from S184 to S201 which are believed to form the transmembrane segment IV were substituted individually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutants exhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined by EPR as a function of the incident microwave power in the presence and absence of two paramagnetic perturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. The periodicity of the sequence-specific variation in the spin-label mobility and the O(2) accessibility parameters unambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier as an alpha-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicating that the helix is amphipatic, with the hydrophilic face containing the residues found to be important for transport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed, as indicated by the values of normalized mobility, which also suggest that the conformational changes occurring during transport probably involve the N-terminal region of the helix.     

Hydrogen exchange and proteolytic degradation of ribonuclease A. Similarities and distinctions of the kinetic mechanisms

The studies by IR spectroscopy of the temperature dependence of the H-D exchange rate of the RNase A peptide NH atoms permit one to characterize two types of conformation fluctuations, local and global. A comparison with the temperature dependence of the proteolytic degradation rate of RNase A shows that similar in nature fluctuations allow for the H-D exchange of NH atoms and the splitting of peptide bonds of the native protein. In the low temperature region, both processes occur through local fluctuations, by way of the EX2 mechanism, and in the high temperature region, they occur through global fluctuations with the overall denaturation desorganization of the native structure, by way of the EX1 mechanism. The biphasic dependence of the rate of H-D exchange and proteolytic degradation of RNase A on urea concentration is also explained by the combination of local and global fluctuations.     

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.     

Lipid-protein interactions in membranes: Effect of lipid composition on mobility of spin-labeled cysteine residues in yeast plasma membrane

In order to gain direct evidence for lipid-dependent protein conformation in membrane, effects of modification of lipid composition on mobility of spin-labeled cysteine residues were investigated in the plasma membrane of the yeast Saccharomyces cerevisiae. Conversion of the bulk of phospholipids to diglycerides by treatment of the membrane with phospholipase C substantially enhanced spectral anisotropy. However, alteration of the viscosity of the lipid-bilayer by enriching the membrane with palmitelaidic or oleic acid had no effect on mobility of spin-labeled cysteine residues. These observations indicate that while the spin-labeled residues are not in direct contact with the lipid core of the membrane, there are lipid-protein interactions to the extent that removal of polar portion of the bulk of phospholipids induces conformational changes in proteins, which in turn restrict mobility of these residues. It is concluded that conformation of membrane proteins depends on lipid structure and that phospholipids have a role in preserving the native conformation of proteins.     

Free cysteine modulates the conformation of human C/EBP homologous protein

The C/EBP Homologous Protein (CHOP) is a nuclear protein that is integral to the unfolded protein response culminating from endoplasmic reticulum stress. Previously, CHOP was shown to comprise extensive disordered regions and to self-associate in solution. In the current study, the intrinsically disordered nature of this protein was characterized further by comprehensive in silico analyses. Using circular dichroism, differential scanning calorimetry and nuclear magnetic resonance, we investigated the global conformation and secondary structure of CHOP and demonstrated, for the first time, that conformational changes in this protein can be induced by the free amino acid L-cysteine. Addition of L-cysteine caused a significant dose-dependent decrease in the protein helicity--dropping from 69.1% to 23.8% in the presence of 1 mM of L-cysteine--and a sequential transition to a more disordered state, unlike that caused by thermal denaturation. Furthermore, the presence of small amounts of free amino acid (80 μM, an 8:1 cysteine∶CHOP ratio) during CHOP thermal denaturation altered the molecular mechanism of its melting process, leading to a complex, multi-step transition. On the other hand, high levels (4 mM) of free L-cysteine seemed to cause a complete loss of rigid cooperatively melting structure. These results suggested a potential regulatory function of L-cysteine which may lead to changes in global conformation of CHOP in response to the cellular redox state and/or endoplasmic reticulum stress.     

Immobilisation of pig liver esterase in hollow fibre membranes

Different methods were evaluated to immobilise Pig Liver Esterase (PLE) in hollow fibre membranes. Four covalent bonding techniques (using epoxy, imidazol, amino and carboxylic acid terminal groups) were tested to link the enzyme to microporous nylon membranes. Physical immobilisation was also studied, by entrapment of the enzyme inside the microporous structure of a polysulfone asymmetric ultrafiltration membrane. The entrapment method lead to a higher retention of enzymatic activity for a longer period of time. This technique was selected to be used in a biphasic membrane bioreactor where the microporous hydrophilic membrane, containing the enzyme, is used to separate an aqueous from an organic phase, in which the substrate is dissolved. Different enzyme loading procedures were studied in the biphasic reactor and the resulting axial and radial enzyme distribution in the hollow fibre module were related to the global enzymatic activity.     

Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex

Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b.     

Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp

All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 cells grown at a low dilution rate in a methanol-limited chemostat culture. The native activator protein (Mr = 50,000) is a dimer of Mr = 27,000 subunits. The N-terminal amino acid sequence revealed no significant similarity with any published sequences. Stimulation of MDH activity by the activator protein required the presence of Mg2+ ions. Plots of specific MDH activity versus activator protein concentration revealed Michaelis-Menten type kinetics. In the presence of activator protein, MDH displayed biphasic kinetics (v versus substrate concentration) toward C1-C4 primary alcohols and NAD. The data suggest that in the presence of activator protein plus Mg2+ ions, MDH possesses a high affinity active site for alcohols and NAD, in addition to an activator- and Mg2(+)-independent low affinity active site. The activation mechanism remains to be elucidated.     

Regulated conformation of myosin V

We have found that myosin V, an important actin-based vesicle transporter, has a folded conformation that is coupled to inhibition of its enzymatic activity in the absence of cargo and Ca(2+). In the absence of Ca(2+) where the actin-activated MgATPase activity is low, purified brain myosin V sediments in the analytical ultracentrifuge at 14 S as opposed to 11 S in the presence of Ca(2+) where the activity is high. At high ionic strength it sediments at 10 S independent of Ca(2+), and its regulation is poor. These data are consistent with myosin V having a compact, inactive conformation in the absence of Ca(2+) and an extended conformation in the presence of Ca(2+) or high ionic strength. Electron microscopy reveals that in the absence of Ca(2+) the heads and tail are both folded to give a triangular shape, very different from the extended appearance of myosin V at high ionic strength. A recombinant myosin V heavy meromyosin fragment that is missing the distal portion of the tail domain is not regulated by calcium and has only a small change in sedimentation coefficient, which is in the opposite direction to that seen with intact myosin V. Electron microscopy shows that its heads are extended even in the absence of calcium. These data suggest that interaction between the motor and cargo binding domains may be a general mechanism for shutting down motor protein activity and thereby regulating the active movement of vesicles in cells.     

Free-energy profiles of membrane insertion of the M2 transmembrane peptide from influenza A virus

The insertion of the M2 transmembrane peptide from influenza A virus into a membrane has been studied with molecular-dynamics simulations. This system is modeled by an atomically detailed peptide interacting with a continuum representation of a membrane bilayer in aqueous solution. We performed replica-exchange molecular-dynamics simulations with umbrella-sampling techniques to characterize the probability distribution and conformation preference of the peptide in the solution, at the membrane interface, and in the membrane. The minimum in the calculated free-energy surface of peptide insertion corresponds to a fully inserted, helical peptide spanning the membrane. The free-energy profile also shows that there is a significant barrier for the peptide to enter into this minimum in a nonhelical conformation. The sequence of the peptide is such that hydrophilic amino acid residues at the ends of the otherwise primarily hydrophobic peptide create a trapped, U-shaped conformation with the hydrophilic residues associated with the aqueous phase and the hydrophobic residues embedded in the membrane. Analysis of the free energy shows that the barrier to insertion is largely enthalpic in nature, whereas the membrane-spanning global minimum is favored by entropy.     

Structured mRNA induces the ribosome into a hyper‐rotated state

During protein synthesis, mRNA and tRNA are moved through the ribosome by the process of translocation. The small diameter of the mRNA entrance tunnel only permits unstructured mRNA to pass through. However, there are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here, we show using single molecule Förster resonance energy transfer and small angle X‐ray scattering experiments a new global conformational state of the ribosome. In the presence of the frameshift inducing dnaX hairpin, the ribosomal subunits are driven into a hyper‐rotated state and the L1 stalk is predominantly in an open conformation. This previously unobserved conformational state provides structural insight into the helicase activity of the ribosome and may have important implications for understanding the mechanism of reading frame maintenance.     

The E. coli replication factor DnaC protein exists in two conformations with different nucleotide binding capabilities. I. Determination of the binding mechanism using ATP and ADP fluorescent analogues

The kinetic mechanism of binding of ATP and ADP fluorescent analogues to the E. coli replicative factor DnaC protein has been studied using the fluorescence stopped-flow technique. The experiments have been performed under pseudo-first-order conditions with respect to the nucleotide cofactor or the DnaC concentration. Three relaxation processes are observed at a large excess of the nucleotide, while only two relaxation processes are detected in the excess of the protein. Such behavior of the kinetic system is a diagnostic indication of the presence of the protein conformational equilibrium prior to the ligand binding. The obtained data indicate that the minimum mechanism that describes the observed kinetics includes the conformational transition of the DnaC protein, prior to nucleotide binding, followed by the two-step, sequential association of the cofactor to only one of the protein conformations, as defined by In the examined solution conditions, the conformation of the DnaC protein is shifted toward the state (DnaC)(2) that binds the nucleotide. The lack of any cofactor binding to the (DnaC)(1) state points to the existence of a stringent locking mechanism of the nucleotide binding-site in the protein. Binding of ATP and ADP analogues obeys the same mechanism, with similar rate constants, indicating that ATP and ADP analogues bind to the same protein conformation. The (C)(1) intermediate dominates the distribution of the DnaC protein population in the presence of cofactors. The formation of (C)(1) is accompanied by a low nucleotide fluorescence increase, indicating a hydrophilic environment around the ribose of bound cofactors. Transition to (C)(2) places the ribose region in a highly hydrophobic environment with relative molar fluorescence intensity approximately 8-fold higher than that of the free cofactor. The significance of these results for the functioning of the DnaC protein is discussed.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of theneu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an α-helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all α-helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Molecular mechanism of recombinant liver fatty acid binding protein's antioxidant activity

Hepatocytes expressing liver fatty acid binding protein (L-FABP) are known to be more resistant to oxidative stress than those devoid of this protein. The mechanism for the observed antioxidant activity is not known. We examined the antioxidant mechanism of a recombinant rat L-FABP in the presence of a hydrophilic (AAPH) or lipophilic (AMVN) free radical generator. Recombinant L-FABP amino acid sequence and its amino acid oxidative products following oxidation were identified by MALDI quadrupole time-of-flight MS after being digested by endoproteinase Glu-C. L-FABP was observed to have better antioxidative activity when free radicals were generated by the hydrophilic generator than by the lipophilic generator. Oxidative modification of L-FABP included up to five methionine oxidative peptide products with a total of ∼80 Da mass shift compared with native L-FABP. Protection against lipid peroxidation of L-FABP after binding with palmitate or α-bromo-palmitate by the AAPH or AMVN free radical generators indicated that ligand binding can partially block antioxidant activity. We conclude that the mechanism of L-FABP''s antioxidant activity is through inactivation of the free radicals by L-FABP''s methionine and cysteine amino acids. Moreover, exposure of the L-FABP binding site further promotes its antioxidant activity. In this manner, L-FABP serves as a hepatocellular antioxidant.     

Functional analysis of a disulfide bond in the cardiac Na(+)-Ca(2+) exchanger

The electrophoretic mobility of the cardiac Na(+)-Ca(2+) exchange protein is different under reducing and nonreducing conditions. This mobility shift is eliminated in a cysteine-less exchanger, suggesting that the presence or absence of an intramolecular disulfide bond alters the conformation and mobility of the exchanger. Using cysteine mutagenesis and biochemical analysis, we have identified the cysteine residues involved in the disulfide bond. Cysteine 792 in loop h of the exchanger forms a disulfide bond with either cysteine 14 or 20 near the NH(2) terminus. Because the NH(2) terminus is extracellular, the data establish that loop h must also be extracellular. A rearrangement of disulfide bonds has previously been implicated in the stimulation of exchange activity by combinations of reducing and oxidizing agents. We have investigated the role of cysteines in the stimulation of the exchanger by the combination of FeSO(4) and dithiothreitol (Fe-DTT). Using the giant excised patch technique, we find that stimulation of the wild type exchanger by Fe-DTT is primarily due to the removal of a Na(+)-dependent inactivation process. Analysis of mutated exchangers, however, indicates that cysteines are not responsible for stimulation of the exchange activity by Fe-DTT. Ca(2+) blocks modification of the exchanger by Fe-DTT. Disulfide bonds are not involved in redox stimulation of the exchanger, and the modification reaction is unknown. Modulation of Na(+)-dependent inactivation may be a general mechanism for regulation of Na(+)-Ca(2+) exchange activity and may have physiological significance.     

1H nuclear-magnetic-resonance studies of the porcine-pancreatic secretory trypsin inhibitor at 270 MHz

The pancreatic secretory trypsin inhibitor from porcine pancreas has been investigated by high-resolution 1H nuclear magnetic resonance (NMR) at 270 MHz. The presence of a number of slowly exchanging labile protons indicates that the protein is highly globular. Of the two tyrosyl rings, one is free-rotating and solvent-exposed while the other one is hindered in its mobility and buried in the interior of the protein. A lineshape analysis of the temperature dependence of aromatic resonances gave the dynamic parameters for activation of ring mobility. The inhibitor exhibits at least three well-resolved high-field ring-current-shifted methyl resonances. Form II of the inhibitor, that lacks the first four residues, has been compared with the intact form I. No detectable differences were found between the spectra of I and II, which indicates that the presence of the N-terminal tetrapeptide does not appreciably affect the overall conformation of the protein.     

Crystal structure of thrombin in a self-inhibited conformation

The activating effect of Na(+) on thrombin is allosteric and depends on the conformational transition from a low activity Na(+)-free (slow) form to a high activity Na(+)-bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na(+) have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na(+) site and the active site regions. Here, we report a 1.87A resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow --> fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10A to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na(+)-binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na(+) and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.     

Exploring the kinetic requirements for enhancement of protein folding rates in the GroEL cavity

The chaperonin system, GroEL and GroES of Escherichia coli enable certain proteins to fold under conditions when spontaneous folding is prohibitively slow as to compete with other non-productive channels such as aggregation. We investigated the plausible mechanisms of GroEL-mediated folding using simple lattice models. In particular, we have investigated protein folding in a confined environment, such as those offered by the GroEL, to decipher whether rate and yield enhancement can occur when the substrate protein is allowed to fold within the cavity of the chaperonins. The GroEL cavity is modeled as a cubic box and a simple bead model is used to represent the substrate chain. We consider three distinct characteristic of the confining environment. First, the cavity is taken to be a passive Anfinsen cage in which the walls merely reduce the available conformation space. We find that at temperatures when the native conformation is stable, the folding rate is retarded in the Anfinsen cage. We then assumed that the interior of the wall is hydrophobic. In this case the folding times exhibit a complex behavior. When the strength of the interaction between the polypeptide chain and the cavity is too strong or too weak we find that the rates of folding are retarded compared to spontaneous folding. There is an optimum range of the interaction strength that enhances the rates. Thus, above this value there is an inverse correlation between the folding rates and the strength of the substrate-cavity interactions. The optimal hydrophobic walls essentially pull the kinetically trapped states which leads to a smoother the energy landscape. It is known that upon addition of ATP and GroES the interior cavity of GroEL offers a hydrophilic-like environment to the substrate protein. In order to mimic this within the context of the dynamic Anfinsen cage model, we allow for changes in the hydrophobicity of the walls of the cavity. The duration for which the walls remain hydrophobic during one cycle of ATP hydrolysis is allowed to vary. These calculations show that frequent cycling of the wall hydrophobicity can dramatically reduce the folding times and increase the yield as well under non-permissive conditions. Examination of the structures of the substrate proteins before and after the change in hydrophobicity indicates that there is global unfolding involved. In addition, it is found that a fraction of the molecules kinetically partition to the native state in accordabce with the iterative annealing mechanism. Thus, frequent "unfoldase" activity of chaperonins leading to global unfolding of the polypeptide chain results in enhancement of the folding rates and yield of the folded protein. We suggest that chaperonin efficiency can be greatly enhanced if the cycling time is reduced. The calculations are used to interpret a few experiments on chaperonin-mediated protein folding.