Biomedical subjects

C R Robinson

Publications and source records attributed to C R Robinson.

At least 19 recordsLinked to original sources

PubMed1998

Optimizing the stability of single-chain proteins by linker length and composition mutagenesis.

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.

Gene Library↗

PubMed1998

Size-dependent increase in prostanoid levels in adenomas of patients with familial adenomatous polyposis.

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.

6-Ketoprostaglandin F1 alpha↗

PubMed1998

Molecular detection of genetic alterations in the serum of colorectal cancer patients.

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.

Alleles↗

PubMed1998

Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease.

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.

Base Composition↗

PubMed1998

Biophysical characterization of the TraY protein of Escherichia coli F factor.

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.

Amino Acid Sequence↗

PubMed1998

A differential scanning calorimetric study of the thermal unfolding of apo- and holo-cytochrome b562.

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.

Apoproteins↗

PubMed1998

Prostaglandin levels in human colorectal mucosa: effects of sulindac in patients with familial adenomatous polyposis.

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.

Adenomatous Polyposis Coli↗

PubMed1997

Energetics of heme binding to native and denatured states of cytochrome b562.

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.

Apoproteins↗

PubMed1997

Formation of a denatured dimer limits the thermal stability of Arc repressor.

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.

Circular Dichroism↗

PubMed1997

Inactivation of Smad4 in gastric carcinomas.

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.

Adenocarcinoma↗

PubMed1997

Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.

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.

Adult↗

PubMed1996

Equilibrium stability and sub-millisecond refolding of a designed single-chain Arc repressor.

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.

Amino Acid Sequence↗

PubMed1996

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

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.

Amino Acid Sequence↗

PubMed1996

Participation of water in Hin recombinase--DNA recognition.

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.

Bacterial Proteins↗

PubMed1995

Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity.

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.

DNA↗

PubMed1995

Hydrostatic and osmotic pressure as tools to study macromolecular recognition.

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.

DNA↗

PubMed1994

Hydrostatic pressure reverses osmotic pressure effects on the specificity of EcoRI-DNA interactions.

To characterize the role of water in protein-DNA interactions, we have studied the specificity of the EcoRI restriction endonuclease as a function of osmotic and hydrostatic pressure. The extent of cleavage by the enzyme at noncanonical ("star") sites is shown to depend uniquely upon the osmotic pressure in the reaction as controlled by the addition of a wide variety of neutral solutes. Alteration of cleavage specificity ("EcoRI* activity") is not uniformly correlated with any other colligative solvent property such as dielectric constant, viscosity, or water concentration. The application of hydrostatic pressure reverses the effects of osmotic pressure, restoring the natural selectivity of the enzyme for its canonical site GAATTC. This combination of observations provides compelling evidence that the site-specific recognition of canonical site DNA by EcoRI is mediated by discretely bound water molecules and that the release of these waters induces a fundamental change in the specificity of the interaction, leading to cleavage at alternative sites. This comprehensive analysis of solvent effects facilitates the unambiguous identification of structurally and functionally specific waters involved in macromolecular recognition events.

DNA↗

PubMed1993

Molecular recognition mediated by bound water. A mechanism for star activity of the restriction endonuclease EcoRI.

Many restriction endonucleases such as EcoRI lose some specificity for their recognition sequence under certain buffer conditions. The cause of this disruption of accurate protein-DNA recognition has never been explained. By cleaving DNA with EcoRI in the presence of several osmolytes, we show that the extent of this EcoRI "star activity" depends strongly upon osmotic pressure. The loss of specificity accompanying decreased water activity implies a role for one or more water molecules in recognition of specific sequences of DNA. Water mediation may constitute a general motif for sequence-specific DNA recognition by restriction enzymes and other DNA-binding proteins.

Binding Sites↗