Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET

Abstract

Fig. 1. Protein-protein interactions between gp32 and gp59 on fDNA. (A) The fluorescence from individual molecules of fDNA with the proteins bound in the order as indicated at the side of each row. The gp32 protein is labeled with A488 (gp32 D ) and the gp59 protein is labeled with A555 (gp59 A ). The filter sets are described in Experimental Methods: F1 is for A488 emission, F2 for FRET between A488 and A555, and F3 for A555 emission. (B) Ensemble FRET studies of Oregon-green-488-maleimide-labeled gp59 titrated into a solution of 400 nM CPM-labeled gp32 and 100 nM fDNA. The fluorescence spectra of 400 nM CPM-gp32 alone (black line), the endpoint of the titration at 1 M Oregon-green-488-maleimide-gp59 (dark gray line), and several intermediate spectra (light gray lines) are shown. (C) Analysis of the donor quenching and acceptor sensitization plotted against the gp59 concentration determines the stoichiometry among gp32, gp59, and fDNA to be 1:1:1 with a calculated binding constant of Ϸ40 nM. The gp32 protein is labeled with A488 (gp32 D ), the gp59 protein is labeled with A555 (gp59 A ), and the gp41 protein is unlabeled. MgATP␥S (500 M) is present for the sample in row 2, and 500 M MgATP is present for the sample in row 3. (B) The gp32 protein is unlabeled, the gp59 protein is labeled with A555 (gp59 A ), and the gp41 is labeled with A488 (gp41 D ). MgATP␥S (500 M) is present for the sample in row 2, and 500 M MgATP is present for the sample in row 3 (5 min after addition of gp41 and nucleotide) and in row 4 (30 min after addition of gp41 and nucleotide). The filter sets are described in the legend to Fig. 1. RNA ͉ tertiary interactions ͉ heterogeneity ͉ time-correlated single photon counting T he discovery that RNAs can catalyze biological reactions has led to intensive effort aimed at identifying additional biological functions for RNA. More than 20 years later, we now know that RNAs play critical functional roles in metabolism, replication, regulation, and development in cells. Extensive biochemical and biophysical studies have led to a better understanding of the molecular mechanisms by which RNAs achieve their biological function, highlighting the important roles of both structure and dynamics. In this regard, single-molecule methods have recently emerged as particularly powerful tools. The folding dynamics of various functional RNAs have been investigated by single-molecule FRET experiments, which probe dynamics under equilibrium conditions via observation of the stochastic fluorescence trajectories as a function of time (1-6). These techniques offer a unique glimpse into subpopulations of a system and, in some cases, have identified conformational heterogeneity or the presence of intermediates that would otherwise be undetectable by ensemble methods (3-5, 7, 8).Unlike the highly cooperative, all-or-none, folding process observed for most protein domains, RNAs generally fold in a noncooperative manner, where the secondary structure forms independently of the tertiary structure. The thermodynamics for f...

Entropic origin of Mg 2+ -facilitated RNA folding

“…Increasing [Mg 2þ ] dramatically accelerates k dock (approximately 12-fold) and slightly decelerates k undock (approximately 1.6-fold) , leading to a steep increase of the docking equi- Fig. 2B), echoing trends observed in an A 7 linked TL-R construct (28). These trends are fit to a four-state kinetic model with Mg 2þ -dependent and -independent docking/undocking pathways, where Mg 2þ association/ dissociation is in rapid equilibrium (Fig.…”

“…1A. Linked by a flexible singlestranded poly(U) junction, the GAAA tetraloop facilely and specifically docks into its receptor, modulating the energy transfer efficiency (E FRET ) between the donor (Cy3) and acceptor (Cy5) fluorophores (16,28). E FRET is monitored by single-molecule confocal microscopy-calculated ratiometrically from the donor and acceptor emission intensities (Materials and Methods) (28,29).…”

“…1A) as a function of [Mg 2þ ]. Dwell times for the TL-R RNA in the docked and undocked states are defined in the time trajectory by crossings of a threshold set at the minimum of the bimodal E FRET distribution (28) (Fig. 1B).…”

“…1B). To achieve a large statistical accuracy in rate constant determination, we calculate probability densities from the cumulative histograms (approximately 30 molecules and >300 transitions) of docked and undocked dwell times under each experimental condition, i.e., Pðτ i Þ ≈ Hðτ i Þ∕½0.5ðτ iþ1 − τ i−1 Þ , where Hðτ i Þ is the histogram value and τ i represents an ordered list of nonzero time bins (28). The normalized probability densities, PðτÞ∕Pð0Þ, are well described by single-exponential decays over >3 orders of magnitude, yielding high quality rate constants, k dock and k undock , from the undocked and docked dwell times, respectively.…”

Laser refrigeration of hydrothermal nanocrystals in physiological media

“…Potential applications for these materials include precision temperature control in integrated electronic (4-6), photonic (7,8), and microfluidic (9) circuits, as well as triggering and probing fundamental metabolic processes (9-11). In particular, the ability to measure and to modulate temperature could enable the investigation of the kinetics and temperature sensitivity of cellular processes, including ion channel actuation (41), conformational folding dynamics of RNA (42), and dynamic stepping motion of molecular myosin (V) motor proteins (43).…”