Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ～10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (～100 μs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.     

Some recent advances in the theory of homogeneous isotropic turbulence

We review some advances in the theory of homogeneous, isotropic turbulence. Our emphasis is on the new insights that have been gained from recent numerical studies of the three-dimensional Navier Stokes equation and simpler shell models for turbulence. In particular, we examine the status of multiscaling corrections to Kolmogorov scaling, extended self similarity, generalized extended self similarity, and non-Gaussian probability distributions for velocity differences and related quantities. We recount our recent proposal of a wave-vector-space version of generalized extended self similarity and show how it allows us to explore an intriguing and apparently universal crossover from inertial- to dissipation-range asymptotics.     

Instrumentation for PSD-based neutron diffractometers at Dhruva reactor

Linear position sensitive detectors (PSDs) are widely used to configure neutron diffractometers and other instruments. Necessary front-end electronics and a data acquisition system [1] is developed to cater to such instruments built around the Dhruva research reactor in BARC. These include three diffractometers with multiple PSDs and four with single PSD. The front-end electronics consists of high voltage units, preamplifiers [2],shaping amplifiers, ratio ADCs (RDC) [3]. The data acquisition system consists of an interface card and software. Commercially available hardware like temperature controller or stepper motor controller connected over GPIB or RS232 are also integrated in the data acquisition system. The data acquisition is automated so that it can continue unattended for control parameter like temperature, thus enabling optimum utilization of available beam time. The instrumentation is scalable and can be easily configured for various instrumental requirements. The front-end electronics and the data acquisition system are described here.     

Spectral studies of some substituted arylthiobenzo[f]quinolines

Synthesis and spectral studies (ir, ¹H nmr, ms) of 1- and 3-substituted arylthiobenzo[f]quinolines (IIIa-IIIc, IVa-IVc) obtained through nucleophilic displacement of the corresponding halobenzo[f]quinoline by various thiols (a-c) were done.     

Red-edge effect in excited state reaction in β-naphthylamine

Alkaline aqueous solutions of β-naphthylamine show the edge effect; a decrease in the relative intensity or emission from the anion (RNH^?) and the neutral molecule (RNH₂) on red-edge excitation (REE) is observed. The nature of quenching of the neutral molecule by OH^? ions also changes on REE. It is suggested that on REE, participation of some non-promoting out-of-plane modes slows down the proton-transfer rate in the excited state.     

Error analysis and efficient sampling in Markovian state models for molecular dynamics

In previous work, we described a Markovian state model (MSM) for analyzing molecular-dynamics trajectories, which involved grouping conformations into states and estimating the transition probabilities between states. In this paper, we analyze the errors in this model caused by finite sampling. We give different methods with various approximations to determine the precision of the reported mean first passage times. These approximations are validated on an 87 state toy Markovian system. In addition, we propose an efficient and practical sampling algorithm that uses these error calculations to build a MSM that has the same precision in mean first passage time values but requires an order of magnitude fewer samples. We also show how these methods can be scaled to large systems using sparse matrix methods.     

Comparison of efficiency and bias of free energies computed by exponential averaging, the Bennett acceptance ratio, and thermodynamic integration

Recent work has demonstrated the Bennett acceptance ratio method is the best asymptotically unbiased method for determining the equilibrium free energy between two end states given work distributions collected from either equilibrium and nonequilibrium data. However, it is still not clear what the practical advantage of this acceptance ratio method is over other common methods in atomistic simulations. In this study, we first review theoretical estimates of the bias and variance of exponential averaging (EXP), thermodynamic integration (TI), and the Bennett acceptance ratio (BAR). In the process, we present a new simple scheme for computing the variance and bias of many estimators, and demonstrate the connections between BAR and the weighted histogram analysis method. Next, a series of analytically solvable toy problems is examined to shed more light on the relative performance in terms of the bias and efficiency of these three methods. Interestingly, it is impossible to conclusively identify a "best" method for calculating the free energy, as each of the three methods performs more efficiently than the others in at least one situation examined in these toy problems. Finally, sample problems of the insertion/deletion of both a Lennard-Jones particle and a much larger molecule in TIP3P water are examined by these three methods. In all tests of atomistic systems, free energies obtained with BAR have significantly lower bias and smaller variance than when using EXP or TI, especially when the overlap in phase space between end states is small. For example, BAR can extract as much information from multiple fast, far-from-equilibrium simulations as from fewer simulations near equilibrium, which EXP cannot. Although TI and sometimes even EXP can be somewhat more efficient in idealized toy problems, in the realistic atomistic situations tested in this paper, BAR is significantly more efficient than all other methods.     

Analytical aspects of some organic acids

Summary A direct complexometric titration of iron(III) with EDTA using o- and p-cresotic acids indicators is given. The colour change at the end point is from blue-violet to light yellow or colourless [at low concentrations of iron(III)]. The optimum range of pH and temperature for carrying out the titrations is 1.99 to 4.2 and 35° to 55°C and 1.42 to 4.4 and 30° to 60° C, respectively. Iron(III) may easily and accurately be estimated in macro- and micro-gram quantities with the help of these metal indicators. Be, Mg, Ca, Sr, Ba, Cd, UO₂ and Mn are without any interference in the estimation. Back titration procedures for determination of zirconium and thorium with iron(III) and o- and p-cresotic acids are carried out.Part V: See Z. analyt. Chem. 173, 196 (1960).