Biomedical subjects

S C Gill

Publications and source records attributed to S C Gill.

10 recordsLinked to original sources

PubMed1994

Modified RNA sequence pools for in vitro selection.

We report the use of modified RNA, in which the 2'-OH group of pyrimidines is replaced by a 2'-amino (2'-NH2) group to identify high affinity ligands specific for human neutrophil elastase (HNE) by in vitro selection. Compared to unmodified RNA the 2'-NH2-modified RNA ligands show enhanced stability in human serum and urine. Use of RNase T1 cleavage data in the presence of K+ and Li+ ions suggests that the modified RNA ligands selected for HNE form an intermolecular G-quartet structure.

Amino Acid Sequence↗

PubMed1994

High-resolution molecular discrimination by RNA.

Species of RNA that bind with high affinity and specificity to the bronchodilator theophylline were identified by selection from an oligonucleotide library. One RNA molecule binds to theophylline with a dissociation constant Kd of 0.1 microM. This binding affinity is 10,000-fold greater than the RNA molecule's affinity for caffeine, which differs from theophylline only by a methyl group at nitrogen atom N-7. Analysis by nuclear magnetic resonance indicates that this RNA molecule undergoes a significant change in its conformation or dynamics upon theophylline binding. Binding studies of compounds chemically related to theophylline have revealed structural features required for the observed binding specificity. These results demonstrate the ability of RNA molecules to exhibit an extremely high degree of ligand recognition and discrimination.

Base Sequence↗

PubMed1994

The solubilities of five cyclic dipeptides in water and in aqueous urea at 298.15 K: a quantitative model for the denaturation of proteins in aqueous urea solutions.

The solubilities of cyclo(L-alanylglycine), cyclo(L-alanyl-L-alanine), cyclo(glycyl-L-leucine), cyclo(L-valyl-L-valine) and cyclo(glycyl-L-phenylalanine) were determined in water and in aqueous urea solutions up to concentrations of 9 molar urea at 298.15 K. The solubilities of all cyclic dipeptides increase with increasing urea concentration. A simple equilibrium model, taking into account the activity of urea and that of water, fits the solubility data yielding apparent equilibrium constants describing the interactions occurring between urea and the peptide groups plus the alkyl groups that are next to these peptide groups. The apparent equilibrium constants were converted to Gibbs energy parameters for each amino acid residue which were then used to make a quantitative estimate of the contribution of urea to the denaturation of proteins.

Chemical Phenomena↗

PubMed1993

Peptide-urea interactions as observed in diketopiperazine-urea cocrystal.

In order to develop a more complete understanding of urea induced protein denaturation we have investigated the crystal structure of urea with the cyclic dipeptide diketopiperazine. This structure, determined to an R factor of 8.1%, shows extensive hydrogen bonding between urea and the peptide groups of diketopiperazine. These studies support a model where hydrogen bonding plays an important contribution in urea-induced protein denaturation. In the companion paper we present thermodynamic data for urea-peptide interactions in aqueous solution that further support this model.

Crystallization↗

PubMed1993

Urea-diketopiperazine interactions: a model for urea induced denaturation of proteins.

The solubility of diketopiperazine (DKP) in aqueous urea (U) solutions with molalities ranging from 0 to 16 mol kg-1 (corresponding to urea activities ranging from 0 to 10 mol kg-1) has been measured as a function of the urea activity at 298.15 K. In accordance with a previous study the solubility of diketopiperazine increases with increasing urea activity but drops sharply at a urea activity of 5.7 +/- 0.2 mol kg-1. This drop in solubility can be attributed to the formation of a DKP.U2 cocrystal. The solubility data were fitted to a simple model based on the stoichiometry of the DKP.U2 to yield an intrinsic equilibrium constant kappa describing the interactions occurring between a urea molecule and a peptide group of diketopiperazine in aqueous solution, its value being kappa = 0.0447 +/- 0.0007 kg mol-1. When the activity of water is taken into account, kappa has a lower value of 0.0398 +/- 0.0007 kg mol-1.

Crystallization↗

PubMed1992

Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer.

To function as a DNA-RNA helicase in rho-dependent transcript termination, six genetically identical subunits of the Escherichia coli transcription termination protein rho must first assemble into a hexameric complex. To help determine the quaternary structure of this complex, we have studied the association equilibria of the rho protomers. Sedimentation equilibrium, sedimentation velocity, diffusion, X-ray scattering, and neutron-scattering data have been combined to create a "phase diagram" of the association states of this protein as a function of protein concentration and ionic environment. The results show that rho exists predominantly as a hexamer under approximately physiological conditions and that this hexamer is in equilibrium with both lower and higher states of association that may also have physiological relevance. Small-angle X-ray scattering measurements and theoretical calculations indicate that the rho hexamer has a radius of gyration of 50 +/- 3 A. The radius of gyration measured by small-angle neutron scattering in 2H2O is 47 +/- 3 A. These scattering studies also support earlier models of rho as a planar hexagon which have been developed on the basis of electron microscopy. In the following paper in this issue [Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C., & von Hippel, P. H. (1992)], these results are combined with information on symmetry, subunit interactions, and packing geometry to obtain a model of the quaternary structure of the functional rho hexamer.

DNA↗

PubMed1991

Escherichia coli sigma 70 and NusA proteins. I. Binding interactions with core RNA polymerase in solution and within the transcription complex.

This paper describes the binding interactions of Escherichia coli transcription factors sigma 70 and NusA with core RNA polymerase, both free in solution and as a part of the functional transcription complex. High pressure liquid chromatography gel filtration and fluorescence techniques have been used to monitor the binding of these factors to core polymerase in solution at salt concentrations roughly comparable to the in vivo environment (250 mM-KCl, 50 mM-potassium phosphate (pH 7.5]; under these conditions all the interacting species exist separately as protein monomers. We find that sigma 70 and NusA binds competitively to core polymerase with a 1:1 binding stoichiometry in this milieu, and that NusA does not bind to the polymerase holoenzyme. Association constants of approximately 2 x 10(9) and 1 x 10(7) M-1 have been measured for the sigma 70-core polymerase interaction and for the NusA-core polymerase interaction, respectively. These findings are consistent with the original formulation of the NusA-sigma 70 cycle put forward by Greenblatt & Li, and provide the basis for a further (and preliminary) quantitative examination of these same interactions within the transcription complex. We use a number of molecular biological techniques, together with data from the literature, to estimate these binding constants in various phases of the transcription cycle. In keeping with our results in solution, we find that the effective binding affinity of sigma 70 for core polymerase within the "open" promoter-polymerase complex is at least 500-fold greater than that of NusA. As the transcription complex moves from the initiation to the elongation phase these relative binding affinities are reversed; the average association constant of NusA for the core polymerase in the elongation complex remains practically the same as in free solution (approx. 3 x 10(7) M-1), while the affinity of sigma 70 for core polymerase in this complex drops to less than 5 x 10(5) M-1. These results are used to begin to define the basic conformational states and interaction potentials of core polymerase in the various stages of the transcription cycle.

Bacterial Proteins↗

PubMed1991

Escherichia coli sigma 70 and NusA proteins. II. Physical properties and self-association states.

In this paper we examine the physical properties and potential for self-association of the Escherichia coli transcription factors, sigma 70 and NusA. We show, by a combination of chemical crosslinking, equilibrium and velocity sedimentation, quasi-elastic light scattering, and small-angle X-ray scattering that NusA exists as a monomer at KCl concentrations between 0.01 and 1.5 M, and that sigma 70 exists as a monomer at KCl concentrations between 0.1 and 1.5 M. The shape and hydration characteristics of each of these monomeric proteins are also examined. The results serve as background for the companion paper in which a thermodynamic analysis is made of the interactions of these transcription factor with E. coli core RNA polymerase in solution and as a component of the functional transcription complex.

Bacterial Proteins↗

PubMed1990

Thermodynamic analysis of the transcription cycle in E. coli.

The E. coli RNA transcription cycle can be divided into three major phases, which are generally called initiation, elongation, and termination. In this paper, we review recent biophysical studies of the interactions of the transcriptional regulatory proteins, sigma 70 and NusA, with themselves and with core RNA polymerase in solution, as well as with core polymerase within the transcription complex. The different affinities of sigma 70 and NusA for core RNA polymerase at various stages in the transcription cycle, together with other quantitative data, are then used to construct a partial free energy diagram for the overall transcription process. This thermodynamic framework, which is interrupted by at least two irreversible steps, can be used to rationalize physiological aspects of the transcription cycle and its regulation, as well as to identify crucial points at which our knowledge is still incomplete.

Bacterial Proteins↗

PubMed1989

Calculation of protein extinction coefficients from amino acid sequence data.

Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.

Amino Acid Sequence↗