Conformational dynamics measurement

R. B. Gregory and A. Rosenberg, Methods Enzymol, 131, 448 (1986). Protein Conformational Dynamics Measured by Hydrogen Isotope Exchange Techniques. 

There has been extensive effort in recent years to use coordinated experimental and simulation studies of polymer melts to better understand the connection between polymer motion and conformational dynamics. Although no experimental method directly measures conformational dynamics, several experimental probes of molecular motion are spatially local or are sensitive to local motions in polymers. Coordinated simulation and experimental studies of local motion in polymers have been conducted for dielectric relaxation,152-158 dynamic neutron scattering,157,159-164 and NMR spin-lattice relaxation.17,152,165-168 A particularly important outcome of these studies is the improved understanding of the relationship between the probed motions of the polymer chains and the underlying conformational dynamics that leads to observed motions. In the following discussion, we will focus on the... 

It puts special emphasis on the rather unique ability of SM FRET measurements to provide information on conformational dynamics and it correlation with conformational structure. 

Thus, whereas ensemble-averaged time-resolved FRET measurements can yield P(R) in an indirect manner, SM FRET measurements can yield P(R) in a direct manner, but only if Tw is very short in comparison to the time scale of conformational dynamics. 

One way of obtaining information on the time scale of conformational dynamics from SM FRET measurements is via the TV-dependence of the... 

Weiss, S. 2000. Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat. Struct. Biol. 7 724-729. 

Beechem J.M. and Haas, E. (1989) Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements, Biophys. J. 55, 1225-1236. 

The conformational dynamics of a biomolecule can be determined by measuring the relaxation properties of the heteronu-clear bond vectors (e.g., the protein backbone bond). 

One can even go so far as to derive the isotherm from dynamic measurements. Note that this does not mean that a dynamic Langmuir isotherm is derived the theory is based on diffusion-limited adsorption, so the surface is taken to be fully relaxed with respect to the sub-surface concentration. In other words, the isotherm is taken to be identical to that in the static case. This is probably correct, unless under dynamic conditions the surfactant assumes a different conformation. 

In another application, 19F NMR studies have provided critical information on the bioactive conformation of taxoids. Fluorine-containing taxoids have been used as probes for NMR analysis of the conformational dynamics of paclitaxel in conjunction with molecular modeling [166], The dependence of the 19F chemical shifts and the Jm--ny values of these fluorinated analogues is examined through 19F and H variable-temperature (VT) NMR measurements. The experiments clearly indicate highly dynamic behavior of these molecules and the existence of equilibrium between conformers. The analysis of the VT NMR data in combination with molecular modeling, including restrained molecular dynamics (RMD), has identified three key conformers, which were further confirmed by the 19F- H heteronuclear NOE measurements. 

The real-time single-turnover trajectories also enabled Xie and coworkers to analyze the time-dependent activity of each enzyme molecule. They found that individual COx molecules show temporal activity fluctuations (i.e., dynamic disorder in activity), attributable to the slow conformational dynamics of the enzyme. The timescale of the activity fluctuation is the timescale of the conformational dynamics that are longer than the catalytic turnovers and can be obtained from the autocorrelation function of the waiting times (Figure 1(d)), which shows an exponential decay behavior versus the index of turnovers (m) and whose decay constant is the fluctuation timescale. This conformational dynamics-coupled enzyme catalysis is fundamental to enzyme catalysis and extremely challenging to study with traditional methods measuring the average behaviors of a population of molecules. 

Time-resolved fluorescence spectroscopy and fluorescence anisotropy measurements have been applied to study (i) excimer formation and energy transfer in solutions of poly(acenaphthalene) (PACE) and poly(2-naphthyl methacrylate) (P2NMA) and (ii) the conformational dynamics of poly(methacrylic acid) (PMA) and poly (acrylic acid) as a function of solution pH. For PACE and P2NMA, analysis of projections in which the spectral, temporal and intensity information are simultaneously displayed have been used to re-examine kinetic models proposed to account for the complex fluorescence decay behaviour that is observed. Time-resolved fluorescence anisotropy measuranents of fluorescent probes incorporated in PMA have led to the proposal of a "connected cluster" model for the hypercoiled conformation of this polymer existing at low pH. 

A further application of time-resolved fluorescence measurements is in the study of conformational dynamics of polymer chains in solution. Fluorescence anisotropy measurements of macromolecules incorporating suitable fluorescent probes can give details of chain mobility and polymer conformation (2,14). A particular example studied in this laboratory is the conformational changes which occur in aqueous solutions of polyelectrolytes as the solution pH is varied (15,16). Poly(methacrylic acid) (PMA) is known to exist in a compact hypercoiled conformation at low pH but undergoes a transition to a more extended conformation at a degree of neutralization (a) of 0.2 to 0.3 (1 6). Similar conformational transitions are known to occur in biopolymer systems and consequently there is considerable interest in understanding the nature of the structures present in model synthetic polyelectrolyte solutions. 

Lane, A. N. and Lefevre, J. F. (1994). Nuclear magnetic resonance measurements of slow conformational dynamics in macromolecules. Methods EnzymoL, 239, 596-619. 

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