ABSTRACT
There is increasing evidence implicating astrocytes in multiple forms of chronic pain, as well as in the specific context of chemotherapy-induced peripheral neuropathy (CIPN). However, it is still unclear what the exact role of astrocytes may be in the context of CIPN. Findings in oxaliplatin and paclitaxel models have displayed altered expression of astrocytic gap junctions and glutamate transporters as means by which astrocytes may contribute to observed behavioral changes. The current study investigated whether these changes were also generalizable to the bortezomib CIPN. Changes in mechanical sensitivity were verified in bortezomib-treated animals, and these changes were prevented by co-treatment with a glial activation inhibitor (minocycline), a gap junction decoupler (carbenoxolone), and by a glutamate transporter upregulator (ceftriaxone). Immunohistochemistry data at day 30 in bortezomib-treated animals showed increases in expression of glial fibrillary acidic protein (GFAP) and connexin 43 but a decrease in GLAST expression. These changes were prevented by co-treatment with minocycline. Follow-up Western blotting data showed a shift in connexin 43 from a non-phosphorylated state to a phosphorylated state, indicating increased trafficking of expressed connexin 43 to the cell membrane. These data suggest that increases in behavioral sensitivity to cutaneous stimuli may be tied to persistent synaptic glutamate resulting from increased calcium flow between spinal astrocytes.
Subject(s)
Astrocytes/metabolism , Bortezomib/toxicity , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2/metabolism , Hyperalgesia/metabolism , Peripheral Nervous System Diseases/metabolism , Spinal Cord/metabolism , Animals , Astrocytes/drug effects , Carbenoxolone/pharmacology , Ceftriaxone/pharmacology , Central Nervous System Agents/pharmacology , Connexin 43/metabolism , Disease Models, Animal , Gap Junctions/metabolism , Glial Fibrillary Acidic Protein/metabolism , Hyperalgesia/pathology , Hyperalgesia/prevention & control , Male , Minocycline/pharmacology , Pain Threshold/drug effects , Pain Threshold/physiology , Peripheral Nervous System Diseases/chemically induced , Peripheral Nervous System Diseases/pathology , Peripheral Nervous System Diseases/prevention & control , Rats, Sprague-Dawley , Spinal Cord/drug effects , Spinal Cord/pathologyABSTRACT
Spinal microglia are widely recognized as activated by and contributing to the generation and maintenance of inflammatory and nerve injury related chronic pain; whereas the role of spinal astrocytes has received much less attention, despite being the first glial cells identified as activated following peripheral nerve injury. Recently it was suggested that microglia do not appear to play a significant role in chemotherapy-induced peripheral neuropathy (CIPN), but in contrast astrocytes appear to have a key role. In spite of the generalizability of astrocyte recruitment across chemotherapy drugs, its correlation to the onset of the behavioral CIPN phenotype has not been determined. The astroglial and microglial markers glial fibrillary acidic protein (GFAP) and OX-42 were imaged here to examine glial reactivity in multiple models of CIPN over time and to contrast this response to that produced in the spinal nerve ligation (SNL) model. Microglia were strongly activated following SNL, but not activated at any of the time points observed following chemotherapy treatments. Astrocytes were activated following both oxaliplatin and bortezomib treatment in a manner that paralleled chemotherapy-evoked behavioral changes. Both the behavioral phenotype and activation of astrocytes were prevented by co-administration of minocycline hydrochloride in both CIPN models, suggesting a common mechanism.
Subject(s)
Antineoplastic Agents/toxicity , Astrocytes/drug effects , Boronic Acids/toxicity , Microglia/drug effects , Organoplatinum Compounds/toxicity , Peripheral Nervous System Diseases/chemically induced , Pyrazines/toxicity , Animals , Astrocytes/pathology , Astrocytes/physiology , Bortezomib , Glial Fibrillary Acidic Protein/metabolism , Hyperalgesia/chemically induced , Hyperalgesia/drug therapy , Hyperalgesia/pathology , Hyperalgesia/physiopathology , Male , Microglia/pathology , Microglia/physiology , Minocycline/pharmacology , Neuroprotective Agents/pharmacology , Oxaliplatin , Peripheral Nervous System Diseases/drug therapy , Peripheral Nervous System Diseases/pathology , Peripheral Nervous System Diseases/physiopathology , Rats, Sprague-Dawley , Spinal Cord/drug effects , Spinal Cord/pathology , Spinal Cord/physiopathology , Spinal Nerves/injuriesABSTRACT
A solvent-exposed Cys11-Cys11' disulfide bond was designed to link the antiparallel strands of the beta sheet both in the Arc repressor dimer and in a single-chain variant in which the Arc subunits are connected by a 15-residue peptide tether. In both proteins, the presence of the disulfide bond increased the T(m) by approximately 40 degrees C. In the single-chain background, the disulfide bond stabilized Arc by 8.5 kcal/mol relative to the reduced form, a significantly larger degree of stabilization than caused by other engineered disulfides and most natural disulfides. This exceptional stabilization arises from a modest effective concentration of the Cys11-Cys11' disulfide in the native state (71 M) and an anomalously low effective concentration in the denatured state (40 microM). Disulfide cross-linking of the two beta strands in the single-chain Arc background accelerated refolding by a factor of 170 into the sub-microsecond time scale. However, the major energetic effect of the disulfide occurs after the transition state for Arc refolding, slowing unfolding by 200 000-fold.
Subject(s)
Bacteriophage P22/genetics , Disulfides/chemical synthesis , Mutagenesis, Insertional , Repressor Proteins/genetics , Repressor Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Disulfides/chemistry , Disulfides/metabolism , Guanidine , Hot Temperature , Hydrogen-Ion Concentration , Models, Chemical , Models, Molecular , Mutagenesis, Insertional/methods , Oxidation-Reduction , Protein Denaturation , Protein Folding , Repressor Proteins/chemical synthesis , Repressor Proteins/chemistry , Thermodynamics , Viral Proteins/chemical synthesis , Viral Proteins/chemistry , Viral Regulatory and Accessory ProteinsABSTRACT
Misfolding and misassembly of proteins are major problems in the biotechnology industry, in biochemical research, and in human disease. Here we describe a novel approach for reversing aggregation and increasing refolding by application of hydrostatic pressure. Using P22 tailspike protein as a model system, intermediates along the aggregation pathway were identified and quantitated by size-exclusion high-performance liquid chromatography (HPLC). Tailspike aggregates were subjected to hydrostatic pressures of 2.4 kbar (35,000 psi). This treatment dissociated the tailspike aggregates and resulted in increased formation of native trimers once pressure was released. Tailspike trimers refolded at these pressures were fully active for formation of infectious viral particles. This technique can facilitate conversion of aggregates to native proteins without addition of chaotropic agents, changes in buffer, or large-scale dilution of reagents required for traditional refolding methods. Our results also indicate that one or more intermediates at the junction between the folding and aggregation pathways is pressure sensitive. This finding supports the hypothesis that specific determinants of recognition exist for protein aggregation, and that these determinants are similar to those involved in folding to the native state. An increased understanding of this specificity should lead to improved refolding methods.
Subject(s)
Glycoside Hydrolases/chemistry , Viral Tail Proteins/chemistry , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Hydrostatic Pressure , Protein Conformation , Protein Folding , Spectrometry, FluorescenceABSTRACT
Recent studies indicate that nonsteroidal anti-inflammatory drugs have a chemopreventive effect against colorectal neoplasia. Nonsteroidal anti-inflammatory drugs inhibit cyclooxygenases, principal enzymes that mediate the formation of prostanoids. To determine whether prostanoids are involved in the pathogenesis of colorectal adenomas, we compared the levels of five major stable metabolic products of the cyclooxygenase pathway in the normal-appearing mucosa and in adenomas of patients with familial adenomatosis polyposis. Of 12 patients tested, 6 had elevated levels of at least one prostanoid in the adenomas. More importantly, the relative levels of three prostanoids [prostaglandin (PG)D2, PGE2, and 6-keto-PGF1alpha] were elevated in adenomas compared to normal-appearing mucosa from the same patients, and the resulting ratios were correlated with the size of the adenoma. These results suggest a role for prostanoids in progression of colorectal polyposis in familial adenomatosis polyposis patients.
Subject(s)
Adenoma/metabolism , Adenomatous Polyposis Coli/metabolism , Prostaglandins/metabolism , 6-Ketoprostaglandin F1 alpha/metabolism , Adenoma/genetics , Adenomatous Polyposis Coli/genetics , Adult , Dinoprost/metabolism , Dinoprostone/metabolism , Female , Humans , Intestinal Mucosa/metabolism , Male , Oxytocics/metabolism , Prostaglandin D2/metabolism , Thromboxane B2/metabolismABSTRACT
Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 microM-10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined. In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.
Subject(s)
Protein Engineering , Protein Folding , Proteins/genetics , Gene Library , Mutagenesis , Proteins/chemistryABSTRACT
Cytochrome b562 is a four-helix-bundle protein containing a non-covalently bound b-type heme prosthetic group. In the absence of heme, cytochrome b562 remains highly structured under native conditions. Here we report thermodynamic data for the thermal denaturation of the holo- and apoproteins as determined by differential scanning calorimetry. Thermal denaturation of holocytochrome b562 is a highly reversible process, and unexpectedly does not involve dissociation of the heme prosthetic group. Thermal denaturation of the corresponding apoprotein, with the heme group chemically removed, remains a cooperative, reversible process. Apocytochrome b562 is substantially destabilized relative to the holoprotein: the t1/2 is more than ten degrees lower, and enthalpy and heat capacity changes are about one-half of the holoprotein values. However, the energetic parameters of apocytochrome b562 denaturation are within the range of observed values for small proteins.
Subject(s)
Cytochrome b Group/chemistry , Escherichia coli Proteins , Escherichia coli/chemistry , Protein Folding , Apoproteins/chemistry , Calorimetry, Differential Scanning , Coenzymes/chemistry , Models, Molecular , Protein Denaturation , Temperature , ThermodynamicsABSTRACT
We have searched for the presence of genetic alterations in serum DNA obtained from 44 colorectal cancer patients. Microsatellite analysis using highly polymorphic markers revealed loss of heterozygosity and/or microsatellite instability in 35 of 44 (80%) primary tumors. No alterations were detected in the paired serum DNA. We next used an oligonucleotide-mediated mismatch ligation assay to detect tumor specific gene mutations in the serum. Among the 16 cases with a K-ras gene mutation in the tumor, the same mutation was detected in three paired serum samples. In the 10 cases with a p53 mutation in the tumor, the identical mutation was detected in seven corresponding serum samples. Comparison of the molecular analysis with clinical diagnosis of these patients revealed that none of the seven Dukes' stage B patients with a K-ras mutation in their tumors demonstrated a mutation in the serum. In contrast, five of seven stage B patients with a p53 mutation in the tumor demonstrated a mutation in the paired serum (P = 0.01, Fisher's exact test). Taken together, either a K-ras or p53 mutation was detected in the serum in 40% of the 25 patients (95% confidence interval, 21-61%), whose primary tumors contained a mutation and in 23% of the 44 patients (95% confidence interval, 12-38%) with colorectal cancer. The frequent detection of p53 mutation in the serum of patients with early stage tumors suggests a possible use of this approach for clinical prognosis and cancer monitoring of colorectal cancer patients.
Subject(s)
Colorectal Neoplasms/blood , Colorectal Neoplasms/genetics , DNA, Neoplasm/blood , DNA, Neoplasm/genetics , Alleles , Chromosome Aberrations , Gene Deletion , Genes, p53 , Genes, ras , Humans , Microsatellite Repeats , Mutation , Prognosis , Sensitivity and SpecificityABSTRACT
Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.
Subject(s)
DNA/chemistry , DNA/metabolism , Deoxyribonuclease EcoRI/metabolism , Base Composition , Base Sequence , Binding Sites , Kinetics , Models, Chemical , Osmotic Pressure , Solutions , Substrate Specificity , ThermodynamicsABSTRACT
Recent evidence suggests that nonsteroidal antiinflammatory drugs (NSAIDs) may prevent colorectal cancer. The mechanism of action of NSAIDs in chemoprevention is unknown but may be linked to their effect on mucosal prostaglandin levels. Levels of five major prostaglandin metabolites were measured by gas chromatography-mass spectrometry in biopsy specimens of flat rectal mucosa from four patients with familial adenomatous polyposis (FAP) before and after sulindac therapy and from five healthy individuals. The prostaglandin present at highest concentration in rectal mucosa from FAP and control subjects was prostaglandin E2. The concentration of thromboxane B2 alone was significantly elevated in FAP patients compared to controls (P = 0.016). In FAP patients treated with sulindac, all prostaglandin metabolite levels were significantly reduced compared to pretreatment levels (P < 0.05) except prostaglandin D2 (P = 0.07). Prostaglandins D2, E2, F2alpha, and 6-keto-F1alpha levels also were significantly reduced in FAP patients on sulindac compared to healthy controls (P < 0.05). However, interpatient heterogeneity of response to sulindac was evident with changes ranging from +19% to -89%, and the patient with the greatest reductions after sulindac developed colorectal cancer after 35 months of therapy. Sulindac treatment, at drug doses shown to regress colorectal adenomas in FAP patients, has heterogeneous effects on the level of major prostaglandins in their rectal mucosa and may not prevent colorectal cancer due to uncoupling of prostaglandin levels and carcinogenesis.
Subject(s)
Adenomatous Polyposis Coli/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Intestinal Mucosa/drug effects , Prostaglandins/analysis , Sulindac/pharmacology , Adenomatous Polyposis Coli/drug therapy , Adult , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Colon , Dinoprost/analysis , Dinoprostone/analysis , Female , Gas Chromatography-Mass Spectrometry , Humans , Intestinal Mucosa/chemistry , Intestinal Mucosa/metabolism , Male , Middle Aged , Prostaglandin D2/analysis , Prostaglandins/metabolism , Prostaglandins F/analysis , Rectum , Sulindac/therapeutic use , Thromboxane B2/analysisABSTRACT
The TraY protein is required for efficient bacterial conjugation by Escherichia coli F factor. TraY has two functional roles: participating in the "relaxosome," a protein-DNA complex that nicks one strand of the F factor plasmid, and up-regulating transcription from the traYI promoter. The traY gene was cloned, and the TraY protein was expressed, purified, and characterized. TraY has a mixed alpha-helix and beta-sheet secondary structure as judged by its circular dichroism spectrum, is monomeric, and undergoes reversible urea denaturation with delta Gu = 6 kcal/mol at 25 degrees C. The kinetics of protein unfolding and refolding, as measured by changes in fluorescence, are complex, suggesting the presence of intermediates or of heterogeneity in the folding reaction. TraY has been classified as a member of the ribbon-helix-helix family of transcription factors but is unusual in appearing to have tandem repeats of the beta alpha alpha motif in the same polypeptide chain. The data presented here show that folding and assembly of the functional (beta alpha alpha)2 unit occurs as an intramolecular reaction and not by cross-folding between different polypeptide chains.
Subject(s)
DNA-Binding Proteins/chemistry , Escherichia coli Proteins , F Factor/chemistry , Amino Acid Sequence , Circular Dichroism , DNA-Binding Proteins/isolation & purification , Escherichia coli , Kinetics , Molecular Sequence Data , Protein Conformation , Protein Denaturation , Protein Folding , Sequence Alignment , Spectrometry, FluorescenceABSTRACT
The thermal stability of the Arc repressor dimer normally increases with concentration because protein folding and subunit association are thermodynamically coupled. At Arc concentrations above 100 microM, however, thermal denaturation remains reversible and cooperative but tm does not continue to increase. In this concentration regime, thermally denatured Arc shows significantly reduced secondary structure and no evidence of a tightly packed core, but light scattering and fluorescence polarization studies indicate that the protein is dimeric. Higher order denatured oligomers are not observed and the stability of the non-native dimer is reduced by Arc mutations, indicating that non-native dimerization involves specific interactions between Arc subunits.
Subject(s)
Repressor Proteins/chemistry , Viral Proteins/chemistry , Circular Dichroism , Dimerization , Heating , Protein Denaturation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Viral Regulatory and Accessory ProteinsABSTRACT
Allelic loss of chromosome 18q has been noted in intestinal type gastric adenocarcinomas. Smad4 is a gene located at 18q that was recently cloned in humans and found to be significantly altered in pancreatic cancers. We sought to determine whether Smad4 genetic alterations played a significant role in gastric tumorigenesis by studying 35 gastric adenocarcinomas of all histopathological types and pathological stages. Microdissected specimens were used for mutational analysis of Smad4 at the nucleotide level, including the entire coding region and intron/exon boundaries. Allelic imbalance was also analyzed at the Smad4 locus using two nearby microsatellite markers. One case of apparent biallelic inactivation of Smad4 was found in our study of 35 gastric carcinomas. A nonsense point mutation at codon 334 was demonstrated, which, similar to other Smad4 mutations, is predicted to truncate the conserved COOH-terminal domain of this protein. This Smad4 C to T transition mutation was proven to be somatically acquired. Allelic loss was also noted on chromosome 18q at a marker near Smad4 in this mutated gastric cancer, apparently producing complete inactivation of Smad4 in this tumor. Significant 18q allelic loss (56% of 34 informative cases) was noted in our gastric carcinomas using microsatellite markers near the Smad4 locus, regardless of histological subtype or pathological stage. Additionally, three cases of microsatellite instability were observed. Thus, Smad4 inactivation was noted in our gastric carcinomas; however, this event was rare. The frequent loss of chromosomal arm 18q observed in gastric cancers suggests the presence of other tumor suppressor genes in this region that are involved in gastric tumorigenesis. Further studies are needed to identify these other targets of inactivation during gastric cancer development.
Subject(s)
Adenocarcinoma/genetics , Chromosomes, Human, Pair 18/genetics , DNA-Binding Proteins , Stomach Neoplasms/genetics , Trans-Activators/deficiency , Alleles , Humans , Loss of Heterozygosity , Microsatellite Repeats , Smad4 Protein , Trans-Activators/geneticsABSTRACT
Aberrant crypt foci (ACF) in colorectal mucosa are proposed to be the earliest morphological lesion in the development of neoplasia, but their characteristics remain controversial. We therefore studied the epithelial phenotype and genotype of ACF from patients with familial adenomatous polyposis (FAP) and of sporadic ACF by evaluating glycoprotein markers associated with neoplasia (lectins Dolichus biflorus agglutinin and peanut agglutinin; monoclonal antibody CA 19-9 against sialyl Lewis-a blood group substance), expression of proliferating cell nuclear antigen, and ras proto-oncogene mutations. The utility of the markers was established by comparing adenomas and hyperplastic polyps. Most FAP ACF resembled adenomas and were found to differ from sporadic ACF in their high frequency of dysplasia, staining with Dolichus biflorus agglutinin, expression of sialyl Lewis-a, proliferation in the epithelium of upper crypts, and low frequency of ras gene mutations (P = .04 to < .0000001). By contrast, sporadic ACF and a subset of FAP ACF had phenotypic characteristics resembling hyperplastic polyps but usually had ras mutations, which were inversely related to dysplasia (P = .00009). Our findings suggest that "aberrant crypt focus" is a generic term analogous to "polyp" and requires further histopathologic, phenotypic, or genotypic classification into dysplastic and heteroplastic (hetero = other, plasia = form) types. Dysplastic ACF represent potential precursors to colorectal adenomas and adenocarcinomas, but heteroplastic ACF appear to be associated, rather than precursor, lesions.
Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Intestinal Mucosa/pathology , Plant Lectins , Adult , Aged , CA-19-9 Antigen , Colonic Polyps/genetics , Colonic Polyps/metabolism , Colonic Polyps/pathology , Colorectal Neoplasms/chemistry , Gangliosides/analysis , Genes, ras , Genotype , Humans , Intestinal Mucosa/chemistry , Lectins , Middle Aged , Mutation , Peanut Agglutinin , Phenotype , Proliferating Cell Nuclear Antigen/analysis , Proto-Oncogene Mas , Staining and LabelingABSTRACT
Cytochrome b562 is a four-helix bundle protein containing a noncovalently bound b-type heme prosthetic group. For the first time, energetics of heme binding to an apocytochrome were measured by isothermal titration calorimetry. The heme is tightly bound to native apocytochrome b562, with a dissociation constant (Kd) of approximately 9 nM (DeltaG degrees = 11 kcal mol-1) at 25 degrees C. Unexpectedly, the thermally denatured apoprotein is also capable of specifically binding heme with modest affinity (Kd = 3 microM, DeltaG degrees = 7.6 kcal mol-1). This interaction results in the dependence of holocytochrome b562 stability on protein concentration in the submicromolar range.
Subject(s)
Cytochrome b Group/chemistry , Cytochrome b Group/metabolism , Escherichia coli Proteins , Escherichia coli/chemistry , Heme/metabolism , Protein Denaturation , Apoproteins/chemistry , Apoproteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Calorimetry , Circular Dichroism , Hemin/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Temperature , ThermodynamicsABSTRACT
Arc-L1-Arc is a single-chain variant of bacteriophage P22 Arc repressor in which a 15 residue linker joins the C-terminus of one subunit to the N-terminus of an otherwise identical subunit. Spectroscopic probes indicate that the native and denatured state of the single-chain protein are similar to those of the unlinked Arc dimer. In equilibrium experiments, Arc-L1-Arc denatures in a reaction without populated intermediate states as judged by the fits of the denaturation isotherms to a two-state model and by the coincidence of denaturation curves monitored by fluorescence and circular dichroism. Comparison of the equilibrium stabilities of Arc-L1-Arc and unlinked Arc gives an effective concentration of subunits in the denatured single-chain variant of 2.7 (+/- 0.7) mM. The kinetic refolding and unfolding reactions of Arc-L1-Arc also appear to proceed without populated intermediates. The rate constant for Arc-L1-Arc unfolding is about 2-fold faster than that of unlinked Arc, indicating that the linker mediates no significant contacts in the native structure that need to be broken to allow unfolding. As expected, the major effect of the linker occurs during the refolding reaction, where the effective subunit concentration calculated from the bimolecular and unimolecular refolding rate constants is 4.5 (+/- 1.8) mM. The transition states for the unfolding and refolding reactions of Arc-L1-Arc and wild-type Arc have similar solvent exposures as measured by the urea dependencies of the equilibrium and rate constants. In the absence of urea, the single-chain protein refolds very rapidly (kf approximately 10(4) s-1) in a reaction that is essentially complete in the sub-millisecond time regime.
Subject(s)
Repressor Proteins/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Bacteriophage P22/chemistry , Bacteriophage P22/genetics , Circular Dichroism , Drug Stability , Escherichia coli/genetics , Kinetics , Models, Molecular , Molecular Structure , Protein Conformation , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Repressor Proteins/genetics , Spectrometry, Fluorescence , Thermodynamics , Viral Proteins/genetics , Viral Regulatory and Accessory ProteinsABSTRACT
The participation of water molecules in the interaction between the Hin recombinase and its operator DNA has been detected by analysis of the dissociation constant in the presence of varying concentrations of neutral solutes and cosolvents. The dissociation constant as measured by gel mobility shift assays increased as the concentration of dimethyl sulfoxide, glycerol, sucrose, or polyethylene glycol was increased. Osmotic pressure is the only property that correlates with the change in the dissociation constant for all compounds. This data indicates that binding of a small population of water molecules accompanies formation of the Hin-DNA complex, and points to a novel role for solvent molecules in assisting site specific interaction between DNA-binding proteins and their cognate DNA sequence.
Subject(s)
Bacterial Proteins/metabolism , DNA Nucleotidyltransferases/metabolism , DNA, Bacterial/metabolism , Peptide Fragments/metabolism , Solvents/pharmacology , Water/pharmacology , Bacterial Proteins/chemistry , DNA Nucleotidyltransferases/chemistry , DNA, Bacterial/chemistry , Dimethyl Sulfoxide/pharmacology , Electrophoresis, Polyacrylamide Gel , Glycerol/pharmacology , Kinetics , Macromolecular Substances , Osmotic Pressure , Peptide Fragments/chemistry , Polyethylene Glycols/pharmacology , Protein Binding/drug effects , Regulatory Sequences, Nucleic Acid , Substrate Specificity , Sucrose/pharmacologyABSTRACT
By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA. Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type. Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration. However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity. This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes. Hence, the activity of the designed Arc-L1-Arc protein is substantially increased relative to wild-type Arc in a variety of assays.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Protein Structure, Secondary , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , Circular Dichroism , Computer Graphics , Drug Stability , Escherichia coli , Kinetics , Macromolecular Substances , Mathematics , Models, Molecular , Models, Theoretical , Molecular Sequence Data , Mutagenesis, Insertional , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repressor Proteins/biosynthesis , Thermodynamics , Viral Proteins/biosynthesis , Viral Regulatory and Accessory ProteinsABSTRACT
The cleavage specificity of the Pvu II and BamHI restriction endonucleases is found to be dramatically reduced at elevated osmotic pressure. Relaxation in specificity of these otherwise highly accurate and specific enzymes, previously termed "star activity," is uniquely correlated with osmotic pressure between 0 and 100 atmospheres. No other colligative solvent property exhibits a uniform correlation with star activity for all of the compounds tested. Application of hydrostatic pressure counteracts the effects of osmotic pressure and restores the natural selectivity of the enzymes for their canonical recognition sequences. These results indicate that water solvation plays an important role in the site-specific recognition of DNA by many restriction enzymes. Osmotic pressure did not induce an analogous effect on the specificity of the EcoRV endonuclease, implying that selective hydration effects do not participate in DNA recognition in this system. Hydrostatic pressure was found to have little effect on the star activity induced by changes in ionic strength, pH, or divalent cation, suggesting that distinct mechanisms may exist for these observed alterations in specificity. Recent evidence has indicated that BamHI and EcoRI share similar structural motifs, while Pvu II and EcoRV belong to a different structural family. Evidently, the use of hydration water to assist in site-specific recognition is a motif neither limited to nor defined by structural families.
Subject(s)
Deoxyribonuclease BamHI/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , DNA/metabolism , Electric Conductivity , Hydrostatic Pressure , Osmotic Pressure , Substrate Specificity , ViscosityABSTRACT
Clearly, hydrostatic and osmotic pressure techniques offer unique potential in the study of fundamental problems of molecular recognition in biological systems. With the recent advances in technology such investigations are rapidly becoming commonplace. We look forward to further advances and their report in succeeding compendiums such as this volume.