Computational investigation of kinetics of cross-linking reactions in proteins: importance in structure prediction

The determination of protein structure using distance constraints is a new and promising field of study. One implementation involves attaching residues of a protein using a cross-linking agent, followed by protease digestion, analysis of the resulting peptides by mass spectroscopy, and finally sequence threading to detect the protein folds. In the present work, we carry out computational modeling of the kinetics of cross-linking reactions in proteins using the master equation approach. The rate constants of the cross-linking reactions are estimated using the pKas and the solvent-accessible surface areas of the residues involved. This model is tested with fibroblast growth factor (FGF) and cytochrome C. It is consistent with the initial experimental rate data for individual lysine residues for cytochrome C. Our model captures all observed cross-links for FGF and almost 90% of the observed cross-links for cytochrome C, although it also predicts cross-links that were not observed experimentally (false positives). However, the analysis of the false positive results is complicated by the fact that experimental detection of cross-links can be difficult and may depend on specific experimental conditions such as pH, ionic strength. Receiver operator characteristic plots showed that our model does a good job in predicting the observed cross-links. Molecular dynamics simulations showed that for cytochrome C, in general, the two lysines come closer for the observed cross-links as compared to the false positive ones. For FGF, no such clear pattern exists. The kinetic model and MD simulation can be used to study proposed cross-linking protocols.     

Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.     

The reactions between formaldehyde,glutaraldehyde and osmium tetroxide,and their fixation effects on bovine serum albumin and on tissue blocks

Summary The reactions between osmium tetroxide and glutaraldehyde and formaldehyde were investigated. It was found that they react together to form intermediate products which then break down to form osmium black. Glutaraldehyde reacts much more rapidly with osmium tetroxide than formaldehyde. The rates of the reactions are increased by increasing the glutaraldehyde concentration or adding bovine serum albumin to the reaction mixture. The reaction rates increase with temperature. The mixtures of fixatives were also tried on tissues and the results paralleled the model experiments. The crosslinking of bovine serum albumin by osmium tetroxide, formaldehyde and glutaraldehyde singly and in mixtures was quantitatively assessed by viscosimetry, gel filtration and disc electrophoresis coupled with densitometry. The crosslinking of bovine serum albumin by pairs of fixatives was less than that produced by the most effective of the pair. After 5 min reaction osmium tetroxide was the most effective crosslinking agent according to viscosimetric experiments, but after one hour's reaction with bovine serum albumin, glutaraldehyde was revealed as the most effective crosslinking agent by gel filtration and electrophoresis.     

Polymerization of proteins with glutaraldehyde. Soluble molecular-weight markers

Glutaraldehyde is well known for its ability to react with proteins and to produce insoluble cross-linked aggregates. In contrast with this situation, conditions are described here which yield covalently linked soluble protein oligomers. The procedure is applicable to a wide range of proteins, and by slight variation in the reaction conditions, soluble polymers in the molecular weight range 3x10(4)-2x10(7) were produced. The products are valuable as molecular-weight markers, e.g. in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The inherent similarities of these oligomers make them superior to commercial molecular-weight protein markers, which may have marked differences in composition and charge.     

Kinesin superfamily proteins and their various functions and dynamics

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.     

An investigation of the co-evolutionary relationships between onchobothriid tapeworms and their elasmobranch hosts

There is general consensus that the living elasmobranchs comprise a monophyletic taxon. There is evidence that, among tetraphyllidean tapeworms, the approximately 201 hooked species (Onchobothriidae) may also comprise a monophyletic group. Determinations of host specificity are contingent upon correct specific identifications. Since 1960, over 200 new elasmobranch species and over 100 new onchobothriid species have been described. Some confidence can be placed in host and parasite identifications of recent studies, but specific identifications provided in older literature in many cases are suspect. There is some consensus among published works on the phylogenetic relationships among elasmobranchs. Phylogenetic relationships among onchobothriids remain largely unresolved. Elasmobranchs have been poorly sampled for onchobothriids; records exist for approximately 20% of the 911 species and approximately 44% of the 170 elasmobranch genera. Onchobothriids are remarkably host specific, exhibiting essentially oioxenous specificity for their definitive hosts. Multiple onchobothriid species commonly parasitise the same host species; in some cases these are congeners, in other cases these are members of two different onchobothriid genera. There is substantial incongruence between available host and parasite phylogenies. For example, Acanthobothrium is by far the most ubiquitous onchobothriid genus, parasitising almost all orders of elasmobranchs known to host onchobothriids, yet, there is no evidence of major clades of Acanthobothrium corresponding to postulated major subgroupings of elasmobranchs (e.g. Galea and Squalea or sharks and rays). Potamotrygonocestus appears to be among the most basal onchobothriid groups, yet it parasitises one of the most derived elasmobranch groups (the freshwater stingray genus Potamotrygon). It appears that congeners parasitising the same host species are not necessarily each other's closest relatives. At this point the preliminary and limited available data suggest that, at least in this system, strict host specificity is not necessarily indicative of strict co-evolution. This study was extremely limited by the lack of available robust phylogenies for onchobothriids and elasmobranchs.     

An extended-X-ray-absorption-fine-structure investigation of diferric transferrins and their iron-binding fragments.

Iron K-edge extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra were recorded for diferric human and rabbit serum transferrins and for diferric chicken ovotransferrin in aqueous solution; for ovotransferrin e.x.a.f.s. spectra from the N-terminal and C-terminal domain fragments were also measured. The overall spectral profiles closely resemble one another, indicating similar iron-binding sites. The simulation of the diferric ovotransferrin spectrum suggests a first co-ordination shell consisting of six low-Z ligands (nitrogen/oxygen), two ligands at a distance of approx. 0.185 nm (1.85 A) and four ligands at approx. 0.204 nm (2.04 A). The two shorter distances may correspond to Fe-O (tyrosine), whereas the longer distance is consistent with Fe-N (histidine) and Fe-O (water). Detailed analysis of the spectra of the N-terminal and C-terminal fragments indicates a difference in the short ligand distance.     

Polarization optical investigation by topo-optical reactions of various blood platelet aggregates

Phases I and II (early and advanced) of platelet aggregation could readily be followed and studied by various topo-optical reactions. Our studies indicate that in the early stage (monitored by an aggregometer) aggregates show no alteration in membrane structure of platelet. In the advanced stage (phase II), in addition to membrane alteration, platelets 2-3 times larger than in the control were observed. These were stained selectively in the pH 1-2 range with thiazine dyes. After hyaluronidase digestion they lost the ability to stain and became birefringent. The elucidation of this phenomenon requires further study.     

Specificity and sequence requirements for interactions between various retroviral Gag proteins.

We previously established a genetic assay for retroviral Gag polyprotein multimerization (J. Luban, K. B. Alin, K. L. Bossolt, T. Humaran, and S. P. Goff, J. Virol. 66:5157-5160, 1992). Here we use this assay to demonstrate homomeric interactions between Gag polyproteins encoded by six different retroviruses. Of the Gag polyproteins tested, only those encoded by closely related retroviruses formed heteromultimers. To determine the primary sequence requirements for human immunodeficiency virus type 1 Gag polyprotein multimerization, we studied the effects on multimerization of deletion and linker insertion mutations. Sequences necessary for this process were located between the C-terminal one-third of the capsid domain and the C terminus of the nucleocapsid domain.     

The kinetics of phosphotriester condensation reactions on solid support.

A study has been made of the kinetics of condensation reactions in phosphotriester synthesis of oligonucleotides on polystyrene copolymer resin support, using monomers, dimers, and trimers as the coupling units. The condensations follow pseudo first order kinetics, with a rate constant that decreases from monomers to dimers to trimers. Arrhenius plots indicate a nearly constant activation energy of around 10.4 kcal mol-1. Extrapolation to find the Arrhenius preexponential factor, A, is inaccurate because of the limited temperature range studied. But if the assumption is made that Ea is the same for monomer, dimer, and trimer reactions, then the A values are inversely proportional to the surface areas of the coupling units, reinforcing the interpretation of A as a collision efficiency factor.     

Upregulation ofE. coli 38kDa proteins induced by glutaraldehyde and formaldehyde

Exposure of the W3110 strain ofEscherichia coli K12 to low concentrations of glutaraldehyde or formaldehyde results in an unusual pattern of protein expression, as determined by high-resolution, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A decline in total protein synthesis is accompanied by the upregulation of three proteins of approximate molecular weight 38 kDa. In the presence of 0.1 mM glutaraldehyde this response occurs within the first 5 min of incubation, and with 0.1 mM formaldehyde, within the first 30 min of incubation. The 38 kDa proteins continue to be expressed at high levels until cell death. Comparison of our 2-D PAGE patterns withE. coli gene-protein and plasmid indexes indicates that one of the proteins may be the major gene product of thepyrC locus. This pattern of protein synthesis may indicate a novelE. coli stress response.