A Quantitative Measurement of Structural changes of RNA kissing complexes using Fluorescence Resonance Energy Transfer

Many RNA interactions in cells occur in the form of loop-loop interactions, also known as a kissing complex . In the bacterial and viral systems discussed here, there are transiently bound proteins involved that modulate the function of kissing complex. These proteins either stabilize the kissing complex or facilitate its conversion to extended duplex. I studied R1inv-R2inv kissing complex (KC), derivatived from RNAI-RNAII complex of E.Coli. Rop protein is known to stabilize the bent R1inv-R2inv KC against dissociation. The goal was to study structural change of this kissing complex after binding of the stabilizing Rop protein. In this work for the first time I used the orientation sensitivity of Fluorescence Resonance energy transfer (FRET) to measure an angular change in the structure of R1inv-R2inv kissing complex upon binding of Rop protein. Single-molecular-pair-FRET (spFRET) is often used to study distance fluctuations of single molecules, it is harder to capture angular changes using FRET, because rotational motion of the dyes tends to wash out the angular sensitivity. The effect of Rop protein on the conformation of the kissing complex is not known. Using fluorescence microscopy techniques we observe a change in twist angle of the KC with protein binding. The eight minimized energy structures reported for R1inv-R2inv KC show a small difference in end-to-end distance and a larger difference in twist and bend angles. From MD simulations I modeled FRET for these eight structures, also for these structures with addition of twist. By comparing the spFRET data with results of this first-principle model, I found the result is consistent with a -25o change in twist angle. My preliminary work on another kissing complex, Dimerization initiation site (DIS) of HIV-1 retrovirus, is also discussed. Nucleocapsid protein (NCp7) plays an important role in facilitating the kissing complex to extended duplex transition for DIS. My work on DIS kissing complex, was aimed at studying possible intermediates in kissing complex to duplex transition, and investigating the effect of proteins like Rop and NCp7. The construction of the TIRF-FRET instrument, methods for surface passivation, and the RNA sequence design are discussed

Single-Molecule-Sensitive FRET in Freely-Diffusing Attoliter Droplets

Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and high-throughput technique that facilitates a substantial increase in signal-to-noise for single-molecular-pair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required.Comment: Twenty-three pages, 3 figures, one table, plus supporting information with additional figures and table

Perbedaan Asupan Lemak, Lingkar Pinggang dan Persentase Lemak Tubuh pada Wanita Dislipidemia dan Non Dislipidemia

Differences of fat intake, waist circumference and percentage of bodt fat in dyslipidemia and non dyslipidemia adult women: Heart disease is the leading cause of death in several countries in the world . One of the major risk factors for heart disease is dyslipidemia . Dyslipidemia is a disorder of lipid metabolism characterized by an increase or decrease in plasma lipid fractions . Dyslipidemia has a strong relationship with the occurrence of central obesity . The purpose of this study was to analyze differences in the intake of fat , waist circumference and body fat percentage in dyslipidemia and non dyslipidemia adult women. This research is analytic study with cross sectional approach . The population in this study were adult women who examined their lipid profile in December 2013 in the Clinical Laboratory Cito Indraprasta Semarang . The total sample was 32 people . Independent test analysis of the differences using t-test for variables waist circumference and Mann Whitney test for variable fat intake and body fat percentage to 95 % and a significance level of 5% error. The results showed 17 adult women ( 53.1 % ) and 15 female adult dyslipidemia ( 46.9 % ) non- dyslipidemia. Average intake of fat, waist circumference and percentage body fat in adult women dyslipidemia higher than non dyslipidemia in adult women. Analysis of statistical tests showed difference in fat intake , waist circumference and body fat percentage in women adult dyslipidemia and non dyslipidemia (p value, respectively p = 0.002, p = 0.0001 and p = 0.0001

The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility

Indocyanine Dyes Approach Free Rotation at the 3′ Terminus of A-RNA: A Comparison with the 5′ Terminus and Consequences for Fluorescence Resonance Energy Transfer

Cyanine dyes are widely used to study the folding and structural transformations of nucleic acids using fluorescence resonance energy transfer (FRET). The extent to which FRET can be used to extract inter- and intramolecular distances has been the subject of considerable debate in the literature; the contribution of dye and linker dynamics to the observed FRET signal is particularly troublesome. We used molecular dynamics (MD) simulations to study the dynamics of the indocarbocyanine dyes Cy3 and Cy5 attached variously to the 3\u27 or 5\u27 terminal bases of a 16-base-pair RNA duplex. We then used Monte Carlo modeling of dye photophysics to predict the results of single-molecule-sensitive FRET measurements of these same molecules. Our results show that the average value of FRET depends on both the terminal base and the linker position. In particular, 3\u27 attached dyes typically explore a wide region of configuration space, and the relative orientation factor, kappa(2), has a distribution that approaches that of free-rotators. This is in contrast to 5\u27 attached dyes, which spend a significant fraction of their time in one or more configurations that are effectively stacked on the ends of the RNA duplex. The presence of distinct dye configurations for 5\u27 attached dyes is consistent with observations, made by others, of multiple fluorescence lifetimes of Cy3 on nucleic acids. Although FRET is frequently used as a molecular ruler to measure intramolecular distances, the unambiguous measurement of distances typically relies on the assumption that the rotational degrees of freedom of the dyes can be averaged out and that the donor lifetime in the absence of the acceptor is a constant. We demonstrate that even for the relatively free 3\u27 attached dyes, the correlation time of kappa(2) is still too long to justify the use of a free-rotation approximation. We further explore the consequences of multiple donor lifetimes on the predicted value of FRET