A minimum-reaction-flux solution to master-equation models of protein folding

Master equations are widely used for modeling protein folding. Here an approximate solution to such master equations is presented. The approach used may be viewed as a discrete variational transition-state theory. The folding rate constant kf is approximated by the outgoing reaction flux J, when the unfolded set of macrostates assumes an equilibrium distribution. Correspondingly the unfolding rate constant ku is calculated as Jpu(1-pu), where pu is the equilibrium fraction of the unfolded state. The dividing surface between the unfolded and folded states is chosen to minimize the reaction flux J. This minimum-reaction-flux surface plays the role of the transition-state ensemble and identifies rate-limiting steps. Test against exact results of master-equation models of Zwanzig [Proc. Natl. Acad. Sci. USA 92, 9801 (1995)] and Munoz et al. [Proc. Natl. Acad. Sci. USA 95, 5872 (1998)] shows that the minimum-reaction-flux solution works well. Macrostates separated by the minimum-reaction-flux surface show a gap in p(fold) values. The approach presented here significantly simplifies the solution of master-equation models and, at the same time, directly yields insight into folding mechanisms.     

Extraction of kinetic information from single-molecule experiments.

We discuss two possible approaches for extracting kinetic information from single-molecule experiments. The first approach is based on computing correlation functions from measured fluorescence signals, and the second on studying the statistics of on and off times of the same fluorescence signal. We show that in both cases it is possible to extract kinetic information about the nature of intramolecular fluctuations of the single molecule. We show that for single-molecule kinetics the intramolecular fluctuations produce stochastic memory effects which lead to new dynamic features that do not exist in traditional chemical kinetics. In particular, we investigate a new type of chemical oscillations in correlation functions observed experimentally by Edman and Rigler (Proc. Natl. Acad. Sci. USA 2000, 97, 8266).     

Mechanisms of selenium-mediated protection from photocarcinogenesis and cell death are not solely p53-dependent

Recent studies published in Oncogene and Proc. Natl. Acad. Sci. USA ascribe a role for selenium, acting through wild type p53, in protecting skin cells in culture from ultraviolet radiation-induced death. While selenium clearly protects cells against ultraviolet radiation-induced death, data that we present and discuss in this letter shows that wild type p53 is not required for such protection. Moreover the non-physiologically high levels of selenium used in some studies leads us to question the relevance of such effects for selenium-induced photoprotection.     

High-pressure transitions in methane hydrate

Three recent studies of the high-pressure transformations of methane hydrate [Chem. Phys. Lett. 325 (2000) 490; Proc. Natl. Acad. Sci. 97 (2000) 13484; Nature 410 (2001) 661] have reported apparently different behaviours. Detailed comparison of our X-ray and neutron diffraction data with those obtained in earlier work shows that there is in fact consistent behaviour on isothermal compression at room temperature with a transition from the clathrate I structure hydrate to a hexagonal hydrate with an unknown structure at 0.9 GPa.     

Denaturing gradient gel electrophoretic analysis of minisatellite alleles

By two-dimensional DNA fingerprinting, an electrophoretic method which combines separation according to size with separation in a denaturing gradient, virtually all minisatellite sequences detected with a minisatellite core probe can be resolved (Uitterlinden et al., Proc. Natl. Acad. Sci. USA 1989, 86, 2742-2746). To investigate the electrophoretic behavior in denaturing gradient gels of allelic restriction fragments containing minisatellite sequences, we analyzed alleles of the two highly polymorphic minisatellite loci D7S22 and D2S44. The results obtained indicate that for these loci, depending on the restriction enzyme used to digest genomic DNA, alleles of different sizes migrate to regions of similar denaturant concentration, i.e. to isothermal positions in the denaturing gradient. Denaturing gradient gel electrophoresis also allows for the discrimination of restriction fragments which are the result of the presence of internal recognition sites in the minisatellite and, therefore, to distinguish between VNTR and restriction site polymorphisms.     

Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting

Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.     

Multiple binding modes of the camptothecin family to DNA oligomers

The binding constants of camptothecin, topotecan and its lactone ring-opened carboxylate derivative to DNA octamers were measured by UV and NMR spectroscopy. The self-association of topotecan (TPT) was also measured. The carboxylate form of TPT binds in the same way as the lactone, but more weakly. Titration of TPT into d(GCGATCGC)2 shows a preferred location stacked onto the terminal G1 base. However, the intermolecular NOEs cannot be reconciled with a single conformation of the complex, and suggest a model of a limited number of conformations in fast exchange. MD calculations on four pairs of starting structures with TPT stacked onto the G1-C8 base pair in different orientations were therefore performed. The use of selected experimental "docking" restraints yielded ten MD trajectories covering a wide conformational space. From a combination of calculated free energies, NOEs and chemical shifts, some of the structures produced could be eliminated, and it is concluded that the data are consistent with two major families of conformations in fast exchange. One of these is the conformation found in a crystal of a TPT/DNA/topoisomerase I ternary complex [Proc. Natl. Acad. Sci. USA 2002, 99, 15 387-15 392].     

Molecular dynamics studies on the interaction of 4-acetylamino-5-hydroxynapthalene-2,7-disulfonic acid with catalytic domain of avian sarcoma virus integrase dimer

We report here 500 ps molecular dynamics (MD) simulation results on two molecules of 4-acetylamino-5-hydroxy naphthalene 2-7-disulfonic acid (Y-3) in the catalytic domain (residues 54–199) of avian sarcoma virus integrase (ASV-IN) dimer. Starting model was obtained on the basis of PDB coordinates (PDB code 1A5X) of ASV-IN-Y3 monomer by Lubkowski et al. [Proc. Natl Acad. Sci. USA, 95 (1998) 4831–4836]. Two molecules of Y3 were docked in the active cavity of the dimer using IMF [Phys. Edu., 5 (1988) 169–176]. Energy minimization (EM) and MD simulation were carried out using Sander's module of [ : Assisted Model building with Energy Refinement: a computer simulation software developed by the University of California, USA, 1997] with all atom force field for Y3 and united atom force field for IN. Analysis of the ligand protein interaction and perturbative changes in the complex is presented in the paper. Salient features related to higher pharmacological activity of Y-3 compared to other analogs from acetyl-amino-naphthalene series are enumerated.     

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity

BACKGROUND: Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake venom alpha-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind alpha-BTX with high affinity, and inhibit its interaction with AChR with an IC(50) of 2 nM. The design of these peptides was based on a lead peptide with an IC(50) of 3x10(-7) M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. RESULTS: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of alpha-BTX with the lead peptide, as well as structure-function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC(50) values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against alpha-BTX lethality. CONCLUSIONS: Synthetic peptides with high affinity to alpha-BTX may be used as potential lead compounds for developing effective antidotes against alpha-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.     

Dynamic models of G-protein coupled receptor dimers: indications of asymmetry in the rhodopsin dimer from molecular dynamics simulations in a POPC bilayer

Based on the growing evidence that G-protein coupled receptors (GPCRs) form homo- and hetero-oligomers, models of GPCR signaling are now considering macromolecular assemblies rather than monomers, with the homo-dimer regarded as the minimal oligomeric arrangement required for functional coupling to the G-protein. The dynamic mechanisms of such signaling assemblies are unknown. To gain some insight into properties of GPCR dimers that may be relevant to functional mechanisms, we study their current structural prototype, rhodopsin. We have carried out nanosecond time-scale molecular dynamics (MD) simulations of a rhodopsin dimer and compared the results to the monomer simulated in the same type of bilayer membrane model composed of an equilibrated unit cell of hydrated palmitoyl-oleoyl-phosphatidyl choline (POPC). The dynamic representation of the homo-dimer reveals the location of structural changes in several regions of the monomeric subunits. These changes appear to be more pronounced at the dimerization interface that had been shown to be involved in the activation process [Proc Natl Acad Sci USA 102:17495, 2005]. The results are consistent with a model of GPCR activation that involves allosteric modulation through a single GPCR subunit per dimer.     

Do-it-yourself microelectrophoresis chips with integrated sample recovery

We present a microelectrophoresis chip that is simple to fabricate using the microfluidic tectonics (microFT) platform (Beebe, D. J. et al., Proc. Natl. Acad. Sci. USA 2000, 97, 13488-13493; Agarwal, A. K. et al.,. J. Micromech. Microeng. 2006, 16, 332-340). The device contains a removable capillary insert (RCI) for easy sample collection after separation (Atencia, J. et al.,. Lab Chip 2006, DOI: 10. 1039/b514068d). Device construction is accomplished in less than 20 min without specialized equipment traditionally associated with microelectrophoresis chip construction. microFT was used to build a PAGE device utilizing two orthogonal microchannels. One channel performs standard separations, while the second channel serves as an access point to remove bands of interest from the chip via the RCI. The RCI contains an integrated electrode that facilitates the removal of bands using electrokinetic techniques. The device was characterized using prestained proteins (Pierce BlueRanger and TriChromRanger). Samples were loaded into the microelectrophoresis device via a standard micropipette. An electrical field of 40 V/cm was used to separate and collect the proteins. The microPAGE device is simple to fabricate, benefits from microscale analysis, and includes an on-chip collection scheme that interfaces the macroworld with the microworld.     

Binding of topotecan to a nicked DNA oligomer in solution

Topotecan (TPT) is in clinical use as an antitumor agent. It acts by binding to the covalent complex formed between nicked DNA and topoisomerase I, and inserts itself into the single-strand nick, thereby inhibiting the religation of the nick and acting as a poison. A crystal structure analysis of the ternary complex has shown how the drug binds (B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, L. Stewart, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15 387-15 392), but has left a number of unanswered questions. Herein, we use NMR spectroscopy and molecular modeling to show that the solution structure of a complex of TPT with nicked natural DNA is similar, but not identical to the crystal conformation, and that other geometries are of very low population. We also show that the lactone form of TPT binds approximately 40 times more strongly than the ring-opened carboxylate.     

Are defect models consistent with the entropy and specific heat of glass formers?

We show that pointlike defect model of glasses cannot explain the thermodynamic properties of glass formers, as for example, the excess specific heat close to the glass transition, contrary to the claim of Garrahan and Chandler [Proc. Natl. Acad. Sci. U.S.A. 100, 9710 (2003)]. More general models and approaches in terms of extended defects are also discussed.     

Inverse Monte Carlo procedure for conformation determination of macromolecules

A novel numerical method for determining the conformational structure of macromolecules is applied to idealized biomacromolecules in solution. The method computes effective inter-residue interaction potentials solely from the corresponding radial distribution functions, such as would be obtained from experimental data. The interaction potentials generate conformational ensembles that reproduce thermodynamic properties of the macromolecule (mean energy and heat capacity) in addition to the target radial distribution functions. As an evaluation of its utility in structure determination, we apply the method to a homopolymer and a heteropolymer model of a three-helix bundle protein [Zhou, Y.; Karplus, M. Proc Natl Acad Sci USA 1997, 94, 14429; Zhou, Y. et al. J Chem Phys 1997, 107, 10691] at various thermodynamic state points, including the ordered globule, disordered globule, and random coil states.     

Electrophoretic purification of the alpha and beta subunits of phosphorylase kinase and evidence in support of the deduced amino acid sequences

A simple, rapid sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method is presented for isolating the alpha, alpha' and beta subunits of rabbit muscle phosphorylase kinase. The SDS-PAGE procedure can yield milligram amounts of alpha and beta from a single preparative gel and also allows isolation of the alpha' isozyme free of alpha. Notably the method provides the purified subunits in a form amenable to structural analysis. Edman degradation of alpha and alpha' reveal identical NH2-terminal structures. Amino acid analysis of the electrophoretically purified alpha and beta subunits are in good agreement with their deduced primary structures. The amino acid sequence of 488 residues in alpha and 713 residues in beta were determined by gas phase Edman degradation. The data support the recently deduced primary structures of alpha (Zander et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 9381-9385).     

Organization of bacteriochlorophylls in individual chlorosomes from Chlorobaculum tepidum studied by 2-dimensional polarization fluorescence microscopy

Chlorosomes are the largest and most efficient natural light-harvesting systems and contain supramolecular assemblies of bacteriochlorophylls that are organized without proteins. Despite a recent structure determination for chlorosomes from Chlorobaculum tepidum (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525), the issue of a possible large structural disorder is still discussed controversially. We have studied individual chlorosomes prepared under very carefully controlled growth condition by a novel 2-dimensional polarization single molecule imaging technique giving polarization information for both fluorescence excitation and emission simultaneously. Contrary to the existing literature data, the polarization degree or modulation depth (M) for both excitation (absorption) and emission (fluorescence) showed extremely narrow distributions. The fluorescence was always highly polarized with M ≈ 0.77, independent of the excitation wavelength. Moreover, the fluorescence spectra of individual chlorosomes were identical within the error limits. These results lead us to conclude that all chlorosomes possess the same type of internal organization in terms of the arrangement of the bacteriochlorophyll c transition dipole moments and their total excitonic transition dipole possess a cylindrical symmetry in agreement with the previously suggested concentric multitubular chlorophyll aggregate organization (Ganapathy Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 8525).