PubMed Health⌕ Search

Biomedical subjects

John F Eccleston

Publications and source records attributed to John F Eccleston.

10 recordsLinked to original sources
PubMed2006

Cooperative hydrogen bond interactions in the streptavidin-biotin system.

The thermodynamic and structural cooperativity between the Ser45- and D128-biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity approximately 2x10(7) times lower than that of wild-type streptavidin at 25 degrees C. The corresponding reduction in binding free energy (DeltaDeltaG) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128-biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128-OD and biotin-N2 is thermodynamically stronger than that between S45-OG and biotin-N1.

Biotin↗
PubMed2006

The kinetic mechanism of the SufC ATPase: the cleavage step is accelerated by SufB.

Protein products of the suf operon are involved in iron-sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')-O-N-methylanthraniloyl-ATP (mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC.mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of Pi. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC.mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and -C proteins interact in the processes involved in regulating iron-sulfur transfer.

Adenosine Diphosphate↗
PubMed2004

Sedimentation equilibrium studies.

The reversible formation of protein-protein interactions plays a crucial role in many biological processes. In order to carry out a thorough quantitative characterization of these interactions, it is essential to establish the oligomerization state of the individual components first. The sedimentation equilibrium method is ideally suited to perform these studies because it allows a reliable, accurate, and absolute value of the solution molecular weight of a macromolecule to be obtained. This technique is independent of the shape of the macromolecule under investigation and allows the determination of equilibrium constants for a monomer-multimer self-associating system.

Animals↗
PubMed2003

The mechanism of Ras GTPase activation by neurofibromin.

Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.

Binding Sites↗
PubMed2003

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct.

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.

Adenosine Diphosphate↗
PubMed2002

H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein.

H-NS plays a role in condensing DNA in the bacterial nucleoid. This 136 amino acid protein comprises two functional domains separated by a flexible linker. High order structures formed by the N-terminal oligomerization domain (residues 1-89) constitute the basis of a protein scaffold that binds DNA via the C-terminal domain. Deletion of residues 57-89 or 64-89 of the oligomerization domain precludes high order structure formation, yielding a discrete dimer. This dimerization event represents the initial event in the formation of high order structure. The dimers thus constitute the basic building block of the protein scaffold. The three-dimensional solution structure of one of these units (residues 1-57) has been determined. Activity of these structural units is demonstrated by a dominant negative effect on high order structure formation on addition to the full length protein. Truncated and site-directed mutant forms of the N-terminal domain of H-NS reveal how the dimeric unit self-associates in a head-to-tail manner and demonstrate the importance of secondary structure in this interaction to form high order structures. A model is presented for the structural basis for DNA packaging in bacterial cells.

Amino Acid Sequence↗
PubMed2002

Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.

Animals↗
PubMed2002

Oligomerization and kinetic mechanism of the dynamin GTPase.

Dynamin is a large molecular weight GTPase. Amongst other biological processes, it is involved in clathrin-dependent endocytosis. It can self-assemble or assemble on other macromolecular structures that result in an increase in its GTPase activity. Its role in endocytosis has been variously attributed to being a force-generating enzyme or a signalling protein. Here we review evidence for the oligomeric state of dynamin at high and low ionic strength conditions. We also review work on the elementary processes of the dynamin GTPase at high ionic strength and compare these to the ATPase of the force-generating protein myosin and the GTPase of the signalling protein Ras. New data on the interaction of dynamin with a fluorescent derivative of GTPgammaS are also presented. The possible mechanism by which assembly of dynamin leads to an increase in its GTPase activity is discussed.

Dynamins↗
PubMed2002

MgF(3)(-) as a transition state analog of phosphoryl transfer.

The formation of complexes between small G proteins and certain of their effectors can be facilitated by aluminum fluorides. Solution studies suggest that magnesium may be able to replace aluminum in such complexes. We have determined the crystal structure of RhoA.GDP bound to RhoGAP in the presence of Mg(2+) and F(-) but without Al(3+). The metallofluoride adopts a trigonal planar arrangement instead of the square planar structure of AlF(4)(-). We have confirmed that these crystals contain magnesium and not aluminum by proton-induced X-ray emission spectroscopy. The structure adopted by GDP.MgF(-) possesses the stereochemistry and approximate charge expected for the transition state. We suggest that MgF3(-) may be the reagent of choice for studying phosphoryl transfer reactions.

Aluminum Compounds↗