Dissecting Ubiquitin Folding Using the Self-Organized Polymer Model

Folding of Ubiquitin (Ub) is investigated at low and neutral pH at different temperatures using simulations of the coarse-grained Self-Organized-Polymer model with side chains. The calculated radius of gyration, showing dramatic variations with pH, is in excellent agreement with scattering experiments. At $T_m$ Ub folds in a two-state manner at low and neutral pH. Clustering analysis of the conformations sampled in equilibrium folding trajectories at $T_m$, with multiple transitions between the folded and unfolded states, show a network of metastable states connecting the native and unfolded states. At low and neutral pH, Ub folds with high probability through a preferred set of conformations resulting in a pH-dependent dominant folding pathway. Folding kinetics reveal that Ub assembly at low pH occurs by multiple pathways involving a combination of nucleation-collapse and diffusion collision mechanism. The mechanism by which Ub folds is dictated by the stability of the key secondary structural elements responsible for establishing long range contacts and collapse of Ub. Nucleation collapse mechanism holds if the stability of these elements are marginal, as would be the case at elevated temperatures. If the lifetimes associated with these structured microdomains are on the order of hundreds of $\mu sec$ then Ub folding follows the diffusion-collision mechanism with intermediates many of which coincide with those found in equilibrium. Folding at neutral pH is a sequential process with a populated intermediate resembling that sampled at equilibrium. The transition state structures, obtained using a $P_{fold}$ analysis, are homogeneous and globular with most of the secondary and tertiary structures being native-like. Many of our findings are not only in agreement with experiments but also provide missing details not resolvable in standard experiments

Mechanistic investigations of pseudouridine synthases : a surprising glycal intermediate lies on the reaction pathway.

Pseudouridine synthases (ΨSs) catalyze the isomerization of uridine (U) in RNA to pseudouridine (Ψ), which is the most common post-transcriptional modification in RNAs and is ubiquitous within all three domains of life. ΨSs are classified into six different families based on sequence alignments and have a universally conserved aspartic acid residue (‘conserved Asp’ for simplicity) that is absolutely essential for activity. RNA containing 5-fluorouridine ([F5U]RNA) has been used as a mechanistic probe. Upon incubation with [F5U]RNA, TruA and RluA get irreversibly inhibited and appear in a protein-RNA adduct band on denaturing PAGE gels, which is consistent with the ‘Michael mechanism’ that proposes a covalent adduct between the conserved Asp and the pyrimidine ring. E. coli TruB does not get inhibited upon incubation with [F5U]RNA, nor does it form an adduct and instead converts F5U into two rearranged, hydrated products. 18O labeling studies with the E. coli ΨSs TruA, TruB, RluA, and TruD show that the hydration of the products of F5U occurs directly from solution and not through the hydrolysis of a Michael adduct. These 18O labeling studies were extended here to Thermotoga maritima TruB (TmTruB) which showed a behavior intermediate between that of E. coli TruB (no observed adduct; multiple turnovers) and RluA and TruA (stoichiometric adduction; single turnover). 18O label is incorporated into RNA directly from solvent during the reaction of [F5U]RNA catalyzed by TmTruB. These results are consistent with the scheme proposed earlier for the handling of F5U by ΨSs. Among examined ΨSs, TmTruB uniquely shows the interesting ability to catalyze the dehydration and rehydration of the products of F5U. The minor product of F5U from the action of ΨSs is an arabino isomer and its generation requires epimerization at C2′, which suggested that the ΨS mechanism may proceed by deprotonation of C2′ to eliminate the uracil and form a glycal intermediate. To test this mechanistic possibility, substrate RNA stem-loops containing [2′-2H]uridine were prepared and used to measure the deuterium kinetic isotope effect on the conversion of U to Ψ. Deuteration at C2′ reduced both Vmax and Vmax/Km for the reaction as catalyzed by TruB (2.54-fold and 3.58-fold, respectively) and RluA (1.79-fold and 2.17-fold, respectively). These results conclusively indicate that the deprotonation of C2′ occurs in a partially rate-determining step in the conversion of U to Ψ, which is consistent with the ‘glycal mechanism’ but inconsistent with the proposed alternatives

A Survey on Issues and Challenges in Congestion Adaptive Routing in Mobile Ad hoc Network

Mobile ad hoc networks is the future wireless communication systems have recently emerged as an important trend. Mobile adhoc network is self-configurable and adaptive. Due to the mobility of nodes, the network congestion occurs and it is difficult to predict load on the network which leads to congestion. Mobile adhoc network suffers from a severe congestion controlling problem due to the nature of shared communication and mobility. Standard TCP controlling mechanism for congestion is not fit to the dynamic changing topology of MANETs. This provides a wide scope of research work in mobile ad hoc network. The purpose of this survey is to study and analyze various issues and challenges in congestion control mechanisms in adaptive routing protocols in Mobile Adhoc Network (MANET)

Propensity to form amyloid fibrils is encoded as excitations in the free energy landscape of monomeric proteins

Protein aggregation, linked to many of diseases, is initiated when monomers access rogue conformations that are poised to form amyloid fibrils. We show, using simulations of src SH3 domain, that mechanical force enhances the population of the aggregation prone ($N^*$) states, which are rarely populated under force free native conditions, but are encoded in the spectrum of native fluctuations. The folding phase diagrams of SH3 as a function of denaturant concentration ($[C]$), mechanical force ($f$), and temperature exhibit an apparent two-state behavior, without revealing the presence of the elusive $N^*$ states. Interestingly, the phase boundaries separating the folded and unfolded states at all [C] and $f$ fall on a master curve, which can can be quantitatively described using an analogy to superconductors in a magnetic field. The free energy profiles as a function of the molecular extension ($R$), which are accessible in pulling experiments, ($R$), reveal the presence of a native-like $N^*$ with a disordered solvent-exposed amino terminal $\beta$-strand. The structure of the $N^*$ state is identical to that found in Fyn SH3 by NMR dispersion experiments. We show that the time scale for fibril formation can be estimated from the population of the $N^*$ state, determined by the free energy gap separating the native structure and the $N^*$ state, a finding that can be used to assess fibril forming tendencies of proteins. The structures of the $N^*$ state are used to show that oligomer formation and likely route to fibrils occur by a domain-swap mechanism in SH3 domain.Comment: 12 pages, 8 figures, 9 supplementary figures (on 5 more pages), 2 supplementary movies (on youtube

Effect of Viscous Dissipation, Soret and Dufour Effect on Free Convection Heat and Mass Transfer from Vertical Surface in a Porous Medium

AbstractIn the present approach, a two dimensional steady free convection flow of heat and mass transfer from a vertical surface in porous media with viscous dissipation has been analyzed numerically considering Soret and Dufour effects. The governing non linear partial differential equations have been transformed by a similar transformation in to a system of ordinary differential equations, which are solved numerically by using implicit finite difference scheme. The dimensionless velocity, temperature and concentration profiles are displayed graphically showing the effects for the different values of the Lewis number, soret number and viscous dissipation parameter