2P107シグナル分子H-Ras活性化の1分子可視化解析 : 生細胞内での局所的な情報伝達のメカニズム

Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.

Single molecule fluorescence resonance energy transfer (FRET) experiments are a powerful and versatile tool for studying conformational motions of single biomolecules. However, the small number of recorded photons typically limits the achieved time resolution. We develop a maximum likelihood theory that uses the full information of the recorded photon arrival times to reconstruct nanometer distance trajectories. In contrast to the conventional, intensity-based approach, our maximum likelihood approach does not suffer from biased a priori distance distributions. Furthermore, by providing probability distributions for the distance, the theory also yields rigorous error bounds. Applied to a burst of 230 photons obtained from a FRET dye pair site-specifically linked to the neural fusion protein syntaxin-1a, the theory enables one to distinguish time-resolved details of millisecond fluctuations from shot noise. From cross validation, an effective diffusion coefficient is also determined from the FRET data.

A few years ago, single molecule Fluorescence Resonance Energy Transfer Scanning Near-Field Optical Microscope (FRET SNOM) images were demonstrated using CdSe semiconductor nanocrystal-dye molecules as donor-acceptor pairs. Corresponding experiments reveal the necessity to exploit much more photostable fluorescent centers for such an imaging technique to become a practically used tool. Here we report the results of our experiments attempting to use nitrogen vacancy (NV) color centers in nanodiamond (ND) crystals, which are claimed to be extremely photostable, for FRET SNOM. All attempts were unsuccessful, and as a plausible explanation we propose the absence (instability) of NV centers lying close enough to the ND border. We also report improvements in SNOM construction that are necessary for single molecule FRET SNOM imaging. In particular, we present the first topographical images of single strand DNA molecules obtained with fiber-based SNOM. The prospects of using rare earth ions in crystals, which are known to be extremely photostable, for single molecule FRET SNOM at room temperature and quantum informatics at liquid helium temperatures, where FRET is a coherent process, are also discussed.

Simultaneous AFM Force Spectroscopy and FRET Measurements on Single Biological Molecules

Dynamic polymorphism of Ras observed by single molecule FRET is the basis for molecular recognition

Single-Molecule Studies of Synaptotagmin and Complexin Binding to the SNARE Complex

2L1600 1分子蛍光共鳴エネルギー移動法によるH-Ras活性化の生細胞内可視化(13.生体膜・人工膜(B)ダイナミクス(E)情報伝達,一般演題,日本生物物理学会第40回年会)

Fret Study of the Binding of HIV-1 RRE RNA to Rev Peptide

We give an overview of the physical basics, experimental techniques, and selected applications of single molecule fluorescence in biophysical research. The photophysical principles underlying single molecule fluorescence resonance energy transfer (smFRET) experiments are treated. Its application in protein biophysics, in particular protein folding and conformational dynamics associated with enzymatic activity, is outlined with selected model systems.

The recent discovery that non-coding RNAs are considerably more abundant and serve a much wider range of critical cellular functions than recognized over previous decades of research into molecular biology has sparked a renewed interest in the study of structure-function relationships of RNA. To perform their functions in the cell, RNAs must dominantly adopt their native conformations, avoiding deep, non-productive kinetic traps that may exist along a frustrated (rugged) folding free energy landscape. Intracellularly, RNAs are synthesized by RNA polymerase and fold co-transcriptionally starting from the 5' end, sometimes with the aid of protein chaperones. By contrast, in the laboratory RNAs are commonly generated by in vitro transcription or chemical synthesis, followed by purification in a manner that includes the use of high concentrations of urea, heat and UV light (for detection), resulting in the denaturation and subsequent refolding of the entire RNA. Recent studies into the nature of heterogeneous RNA populations resulting from this process have underscored the need for non-denaturing (native) purification methods that maintain the co-transcriptional fold of an RNA. Here, we present protocols for the native purification of an RNA after its in vitro transcription and for fluorophore and biotin labeling methods designed to preserve its native conformation for use in single molecule fluorescence resonance energy transfer (smFRET) inquiries into its structure and function. Finally, we present methods for taking smFRET data and for analyzing them, as well as a description of plausible overall preparation schemes for the plethora of non-coding RNAs.

Single Molecule Study Of the RNA Degradation and Polyadenylation Activities of the Archaeal Exosome

Single Molecule Studies of the c-di-GMP Riboswitch

Single Molecule Views of the Ribosome Assembly

Single Molecule FRET Study of Nucleotide Binding Effects on Muts Proteins

Conformational Changes in MutS during Mismtach Repair Signaling Determined with Single Molecule FRET