Biomedical subjects

R R Koepsel

Publications and source records attributed to R R Koepsel.

18 recordsLinked to original sources

PubMed2003

A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis.

A Windows program for metabolic engineering analysis and experimental design has been developed. A graphical user interface enables the pictorial, "on-screen" construction of a metabolic network. Once a model is composed, balance equations are automatically generated. Model construction, modification and information exchange between different users is thus considerably simplified. For a given model, the program can then be used to predict all the extreme point flux distributions that optimize an objective function while satisfying balances and constraints by using a depth-first search strategy. One can also find the minimum reaction set that satisfies different conditions. Based on the identified flux distributions or linear combinations, the user can simulate the NMR and GC/MS spectra of selected signal molecules. Alternately, spectra vectorization allows for the automated optimization of labeling experiments that are intended to distinguish between different, yet plausible flux extreme point distributions. The example provided entails predicting the flux distributions associated with deleting pyruvate kinase and designing 13C NMR experiments that can maximally discriminate between the flux distributions.

Computer Simulation↗

PubMed2000

Coordination of two high-affinity hexamer peptides to copper(II) and palladium(II) models of the peptide-metal chelation site on IMAC resins.

The coordination of peptides Ser-Pro-His-His-Gly-Gly (SPHHGG) and (His)6 (HHHHHH) to [PdII(mida)(D2O)] (mida2- = N-methyliminodiacetate) was studied by 1H NMR as model reactions for CuII(iminodiacetate)-immobilized metal affinity chromatography (IMAC) sites. This is the first direct physical description of peptide coordination for IMAC. A three-site coordination is observed which involves the first, third, and fourth residues along the peptide chain. The presence of proline in position 2 of SPHHGG achieves the best molecular mechanics and bonding angles in the coordinated peptide and enhances the interaction of the serine amino nitrogen. Histidine coordination of H1, H3, and H4 of (His)6 and H3 and H4 of SPHHGG was detected by 1H NMR contact shifts and H/D exchange of histidyl protons. The EPR spectra of SPHHGG and HHHHHH attached to the [CuII(mida)] unit were obtained for additional modeling of IMAC sites. EPR parameters of the parent [Cu(mida)(H2O)2] complex are representative: gzz = 2.31; gyy = 2.086; gxx = 2.053; A parallel = 161G; AN = 19G (three line, one N coupling). Increased rhombic distortion is detected relative to the starting aqua complex in the order of [Cu(mida)L] for distortion of HHHHHH > SPHHGG > (H2O)2. The lowering of symmetry is also seen in the decrease in the N-shf coupling, presumably to the imino nitrogen of mida2- in the order 19 G (H2O), 16 G (SPHHGG) and 11 G (HHHHHH). Visible spectra of the [Cu(mida)(SPHHGG)] and [Cu(mida)(HHHHHH)] as a function of pH indicate coordination of one histidyl donor at ca. 4.5, two in the range of pH 5-7, and two chelate ring attachments involving the terminal amino donor for SPHHGG or another histidyl donor of HHHHHH in the pH domain of 7-8 in agreement with the [PdII(mida)L] derivatives which form the two-chelate-ring attachment even at lower pH as shown by the 1H NMR methods.

Catalysis↗

PubMed2000

Growth factor and cytokine gene expression in mechanically strained human osteoblast-like cells: implications for distraction osteogenesis.

OBJECTIVE: An understanding of bone cellular biology is a basic necessity to understanding events such as distraction osteogenesis. The goal of this study was to determine the effect of continuous cyclic mechanical stretch as a fundamental event in distraction osteogenesis on the expression of 3 bone growth factors, transforming growth factor-beta 1 (TGF-beta1), insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF) and 2 cytokines, interleukin (IL)-1 (IL-1) and 6 (IL-6) in human osteoblast-like cells. MATERIAL AND METHODS: A human osteoblast-like cell line, SaOS-2, capable of forming a ground substance and mineralizing it, was maintained. Cells were transferred to 6-well plates with flexible silicon bottoms grown to confluence and either subjected to tensile stretch for different time intervals or used as the control group. RNA was isolated to conduct Northern blot analysis for the expression of 3 bone growth factors, transforming TGF-beta1, IGF-1, bFGF, and 2 cytokines, IL-1 and IL-6. RESULTS: After 8 hours, mRNA for TGF-beta1 and IGF-1 increased in the experimental group, whereas bFGF decreased but cytokines IL-1 and IL-6 were not affected. At 16 hours, TGF-beta1, IGF-1, and bFGF showed increased levels of mRNA; IL-6 showed a slight increase. After 24 hours, TGF-beta1, IGF-1, bFGF, and IL-6 had increased mRNA levels. IL-1beta did never show significant alterations in mRNA production as compared with the control. CONCLUSION: Tensile stretch on osteoblast-like cells alter local regulation of bone formation, increasing the expression of bone growth factors, whereas catabolic cytokines are unaffected. These findings suggest a direct effect of mechanical strain on osteoblasts and may be the driving factors of bone growth during distraction.

Blotting, Northern↗

PubMed2000

Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant.

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Bacillus subtilis↗

PubMed1999

Vector engineering anomalies: impact on fusion protein purification performance.

Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags. We have identified several tags useful for protein purification on Zn(II)-IDA columns. These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5. Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon. The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column. Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein.

Chromatography, Affinity↗

PubMed1997

Selection of optimum affinity tags from a phage-displayed peptide library. Application to immobilized copper(II) affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a versatile tool for the purification of proteins with affinity for immobilized metals. Moreover, this technique has also been used for the separation of proteins that do not exhibit significant metal affinity in the native form, by their fusion to a short metal-binding peptide (a tail), most commonly, a sequence consisting of six adjacent histidine residues (His6). A phage-displayed random hexamer library is used to select for peptides with affinity for immobilized copper. The study follows our previous investigation in which a stringent selection protocol led to the selection of only one copper-binding peptide containing two histidines. The less stringent conditions employed in this work resulted in the selection of a more diverse population of peptides, but again, dominated by peptides containing two histidines (13 out of 19). The prevalence of peptides with two histidines, in contrast to peptides with a higher number of histidines (e.g. His6 or HHHMVH), is explained based on the differences in the pH dependence of their affinity for copper. As discussed, the selected peptides with two histidines will be superior affinity tails than peptides with a higher histidine content (e.g. His6). Moreover, a peptide with a single histidine but with a very high copper affinity, is also identified. Its high copper affinity is related to the presence of several hydrophobic residues in the neighborhood of histidine. Chromatography of human interleukin-1 beta (hIL-1 beta) and several other proteins containing a single surface-exposed histidine surrounded by several hydrophobic residues confirmed that such a sequence could also serve as a very effective metal binding domain for protein purification using immobilized copper(II) columns.

Affinity Labels↗

PubMed1997

Identification of zinc proteins in rat parotid saliva.

The presence of unique zinc-binding proteins in human saliva is well documented. These observations have not, however, been extended to other species. The rat has been used extensively to study the salivary gland and its secretion, and it is therefore important to determine if the spectrum of zinc-binding proteins in this experimental model resembles that found in humans. To begin the analysis of zinc-binding proteins in stimulated rat parotid saliva, the saliva was fractionated by DEAE Sephadex and Sepharose 6B chaelate chromatography and the protein patterns analysed by electrophoresis. Zinc-binding proteins from the parotid saliva were identified by incubating Western blots with 65Zn and identifying any bound zinc by autoradiography. Comparison of the autoradiograms with the Coomassie blue-stained filter revealed several proteins with zinc-binding capacity. Isolation of the major zinc-binding proteins revealed an amino acid composition of proline 28%, glutamine 19% and glycine 15%, which is consistent with the amino acid composition of rat salivary acidic proline-rich protein. In addition to the proline-rich proteins, one other zinc-binding protein was analysed. The N-terminal sequence of this protein was found to bear a striking similarity (16 out of 20 amino acids) to secreted carbonic anhydrase VI of the mouse, a known zinc-binding protein. These data demonstrate that rat acidic proline-rich proteins, having an amino acid composition similar to that in humans, have zinc-binding potential. The data also confirm previous reports suggesting secreted carbonic anhydrase in rat parotid saliva.

Amino Acid Sequence↗

PubMed1993

Structural basis for the sequence selectivity of DNA cleavage by bleomycins.

DNA strand scission by the bleomycin analogs talisomycin S10b and a novel fluorescent mimic, fluoromycin, has been characterized and compared with that of bleomycin A2. Both analogs were found to have essentially the same sequence specificity for DNA strand scission as bleomycin A2. As observed with bleomycin A2, the preferred sites of DNA cleavage by talisomycin S10b and fluoromycin were 5'-GpT-3' and 5'-GpC-3'. These results suggest that the DNA sequence selectivity of bleomycins remains unaltered upon modification of the C-terminal domain.

Base Sequence↗

PubMed1989

Synthesis of single-stranded plasmid pT181 DNA in vitro. Initiation and termination of DNA replication.

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.

Blotting, Southern↗

PubMed1988

Mechanism of plasmid pT181 DNA replication.

The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. This nick presumably serves as the start-site of pT181 replication by extension synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. In vitro replication of a recombinant plasmid carrying two pT181 origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading-strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two PT181 origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.

Bacterial Proteins↗

PubMed1987

Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.

RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities. These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism. RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form. We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication. We have mapped the secondary cleavage sites on pT181 DNA. When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved. However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed. The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site. The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage.

Bacterial Proteins↗

PubMed1986

Static and initiator protein-enhanced bending of DNA at a replication origin.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, DNA packaging, and the recognition of specific DNA sequences by proteins. It has recently been demonstrated that DNA bending can be sequence-directed. Bent DNA has also been observed as a consequence of sequence-specific binding of proteins to DNA. In this report DNA of plasmid pT181 is shown to contain a bend at the replication origin. Furthermore, this bend is enhanced by the binding of the pT181 replication initiator protein, RepC, to the origin.

Bacterial Proteins↗

PubMed1986

Sequence-specific interaction between the replication initiator protein of plasmid pT181 and its origin of replication.

The replication of the pT181 plasmid is dependent on the plasmid-encoded initiator protein RepC. We have previously shown that RepC protein has sequence-specific endonuclease and topoisomerase-like activities. In this paper we demonstrate that this initiator protein has sequence-specific DNA-binding properties. Based on filter binding of plasmid restriction fragments, RepC protein specifically recognizes only the pT181 origin region. Using DNase I and neocarzinostatin "footprinting" techniques, we show that RepC protein specifically binds to a 32-base-pair sequence within the origin that is part of the initiator cistron. Using dimethyl sulfate as a chemical probe, we have identified the purine residues that interact with the initiator protein. The features of the DNA region that interacts with RepC protein include sequences with the potential to form Z DNA and/or hairpin structures. The specific DNA-protein interaction at the origin may be critical in the initiation of pT181 DNA replication by RepC protein in association with other host initiation proteins.

Bacterial Proteins↗

PubMed1985

Purification of pT181-encoded repC protein required for the initiation of plasmid replication.

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 replication specifically requires the plasmid-encoded repC protein. An in vitro system has been shown to carry out semiconservative replication of pT181 and its derivative plasmids (Khan, S A., Carleton, S. M., and Novick, R. P. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4902-4906). We have used this replication assay to isolate repC protein, which was purified to near homogeneity. The repC gene was cloned into the pKJB825 plasmid that contains the phage lambda temperature-sensitive repressor gene, cI857, and the rightward promoter, PR. Upon temperature induction, Escherichia coli clones containing the recombinant plasmid overproduced repC protein, which was purified in significant quantities. The molecular weight of repC protein under denaturing conditions is 38,000, which is consistent with the size predicted from the DNA sequence data. Presence of repC protein was absolutely essential for the initiation of replication of pT181 and its derivatives in vitro.

Bacterial Proteins↗

PubMed1985

The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities.

Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein. Here we demonstrate that highly purified RepC protein has sequence-specific endonuclease and topoisomerase-like activities. A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking-closing by RepC protein. RepC introduces a single strand break within the pT181 origin. The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence. This nick site probably corresponds to the start site of pT181 replication. The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism.

Bacterial Proteins↗

PubMedDate not supplied

Comparison of energy-minimized structures of [Pd(II)(N-methyliminodiacetate)] complexes of X(1)-His-X(3)-His-His peptides as an analysis of steric and specific interactions with synthetic binding tags for IMAC separations.

[Pd(II)(mida)(peptide)] complexes for the series of peptides of sequence X(1)-His-X(3)-His-His were studied by molecular mechanics methods using Spartan, MMFF94, and SYBYL programs with X(1) = X(3) = glycine (G), phenylalanine (F), tyrosine (Y), tryptophan (W), and with X(1) = glycine (G) and X(3) = proline (P). For comparison purposes, data were also obtained for the Ser-Pro-His-His-Gly (SPHHG) and the (His)(5) peptides.The latter two peptides and GHPHH are tags in current use for IMAC separations. These provide calibration points as to the binding affinities that have been determined for the entire series. The energies of the complexes, as an average trend found from the composite behavior of the three methods, were found to be SPHHG (205 kcal/mol) (most stable; are values obtained by MMFF94 methods) < HH(#)HH(#)H(#) (222; where # implies the site of attachment to match the other X(1)-His-X(3)-His-His peptides) < YHYHH (249) < GHGHH (265) < WHWHH (284) approximately GHPHH (286) < FHFHH (311) (least stable), implying that FHFHH might be a useful chromatographic tag for IMAC protein separations that would elute more readily than GHPHH from IMAC sites that are of square-planar structure, such as Cu(II)(ida-supported) IMAC columns. Specific H-bonded interactions are observed between the tyrosine X(1) and pendant carboxylates and between X(3) and the N-terminal amine of [Pd(mida)(YHYHH)]. Face-to-pi-face ring stacking occurs between phenylalanine X(1) and X(3) units in [Pd(mida)(FHFHH)], whereas edge C-H to pi H-bonding or pi stacking occurs between the X(1) and X(3) tryptophans of [Pd(mida)(WHWHH)]. Two energy minima were found with tryptophan. The more stable form has the aromatic rings more parallel, similar to the stacked form of phenylalanine, rather than the edge C-H to pi H-bonding, and virtually the same overall energy as for [Pd(mida)(GHPHH)]. The "perpendicular" structure was found as an initial local energy minimum, but additional MMFF94 calculations found the pi -stacked arrangement at energy ca. 39 kcal/mol lower than that of the nearly "perpendicular" arrangement of the tryptophan rings, a composite effect of relaxation of the peptide, together with differences in stabilities imparted by the differing geometries. The use of the terms "pi-stacked " and "perpendicular" forms represent the limiting cases available to the tryptophan side chain groups. A twist of about 15 degrees to 20 degrees in dihedral angle is all that is necessary to change between structures that are nearly described as one form or the other.

Chromatography, Affinity↗

PubMedDate not supplied

Cell growth and by-product formation in a pyruvate kinase mutant of E. coli.

In this paper, we report on the analysis of acid formation in an E. coli pyk mutant. The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations. However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type. This substantial reduction in acids is accompanied by an increase in CO(2) production. Moreover, unlike the B. subtilis pyk mutant, the E. coli pyk mutant did not show a substantial increase in the PEP pool.

Acids↗

PubMedDate not supplied

Design of affinity tags for one-step protein purification from immobilized zinc columns.

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Affinity Labels↗