Article Information
PubMed
Related Articles
- Conformational Selection During Weak Binding at the Actin an...Conformational Selection During Weak Binding at the Actin and Myosin Interface
Biophysical Journal, Volume 79, Issue 3, 1 September 2000, Pages 1498-1510
Jin Xu and Douglas D. Root
AbstractThe molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the regulatory light chain in the presence of nucleotide transition state analogs. Only small changes (<0.5nm) were detected that are consistent with internal conformational changes of the myosin molecule, but not with extreme differences in the average lever arm position suggested by some atomic models. These results were confirmed by stopped-flow resonance energy transfer measurements during single ATP turnovers on myosin. To examine the participation of actin in the powerstroke process, resonance energy transfer between the regulatory light chain on myosin subfragment-1 and the C-terminus of actin was measured in the presence of nucleotide transition state analogs. The efficiency of energy transfer was much greater in the presence of ADP-AlF, ADP-BeF, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached cross-bridges that appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin.
Abstract | Full Text | PDF (412 kb) - In Vivo Measurement of Intramolecular Distances Using Geneti...In Vivo Measurement of Intramolecular Distances Using Genetically Encoded Reporters
Biophysical Journal, Volume 93, Issue 9, 1 November 2007, Pages L45-L47
Walter Sandtner, Francisco Bezanilla and Ana M. Correa
AbstractThe function of membrane proteins occurs in the context of the cell membrane in living cells acting in concert with various cell components such as other proteins, cofactors, etc. The understanding of the function at the molecular level requires structural techniques, but high resolution structural studies are normally obtained in vitro and in artificial membranes or detergent. Ideally the correlation of structure and function should be carried out in the native environment but most of the techniques applicable in vivo lack the high resolution necessary to track conformational changes on a molecular level. Here we report on the successful application of an improved variant of lanthanide-based resonance energy transfer a fluorescent based technique, to potassium channels expressed in live oocytes. Lanthanide-based resonance energy transfer is particularly suitable to measure intramolecular distances with high resolution. The improvements reported in this work are mainly based on the use of two different small genetically encoded tags (the Lanthanide Binding Tag and the hexa-histidine tag), which due to their small size can be encoded at will in many positions of interest without distorting the protein's function. The technique reported here has the additional improvement that the two tags can be placed independently in contrast to previously described techniques that rely on chemical labeling procedures of thiols.
Abstract | Full Text | PDF (211 kb) - Temporally and Spectrally Resolved Imaging Microscopy of Lan...Temporally and Spectrally Resolved Imaging Microscopy of Lanthanide Chelates
Biophysical Journal, Volume 74, Issue 5, 1 May 1998, Pages 2210-2222
György Vereb, Elizabeth Jares-Erijman, Paul R. Selvin and Thomas M. Jovin
AbstractThe combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope.
Abstract | Full Text | PDF (797 kb) - …more
Copyright © 1998 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 74, Issue 5, 2451-2458, 1 May 1998
doi:10.1016/S0006-3495(98)77953-6
Luminescence Resonance Energy Transfer Measurements in Myosin
Elise Burmeister Getz*, #, Roger Cooke# and Paul R. Selvin*, §, 
, 
* Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720 USA
# Department of Biochemistry and Biophysics and the Cardiovascular Research Institute, University of California, San Francisco, California 94143 USA
§ Department of Physics, University of Illinois, Urbana, Illinois 61801 USA
Address reprint requests to Dr. Paul Selvin, Loomis Laboratory of Physics, 1110 W. Green St., University of Illinois, Urbana, IL 61801. Tel.: 217-244-3371; Fax: 217-244-7187.Abstract
Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), which uses a luminescent lanthanide as the energy-transfer donor, is capable of measuring these long distances. Specifically, we measure distances between the catalytic domain (Cys707) and regulatory light chain domain (Cys108) of the myosin head. An energy transfer efficiency of 21.2±1.9% is measured in the myosin complex without nucleotide or actin, corresponding to a distance of 73Å, consistent with the crystal structure of Rayment et al. Upon binding to actin, the energy transfer efficiency decreases by 4.5±1.0%, indicating a conformational change in myosin that involves a relative rotation and/or translation of Cys707 relative to the light chain domain. Addition of ADP also alters the energy transfer efficiency, likely through a rotation of the probe attached to Cys707. These results demonstrate that LRET is capable of making accurate measurements on the relatively large actomyosin complex, and is capable of detecting conformational changes between the catalytic and light chain domains of myosin.
