Name: demonstrating the uses of the novel gravitational force spectrometer to stretch and measure fibrous proteins
Uploaded: 2011-03-19T11:00:00
Description: Watch this Scientific Journal Video about Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins at JoVE.com

This material is based upon work supported by the National Science Foundation under Grant No. 0842736.

Introduction to the Novel GFS Configuration
 The GFS consists of several essential components: A regular light microscope, an equatorial mount, a camera, and a computer [Figure 1]. The sealed flow-cell chamber which holds the sample is also indispensible according to the GFS design. The Light microscope is mounted onto the equatorial mount so the scope can be rotated into different orientations in space. This ability allows the static vector of gravity to be exploited so that the samples can be ...

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	<li>Schwaiger, I., Sattler, C., Hostetter, D. R., Rief, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myosin+coiled-coil+is+a+truly+elastic+protein+structure.">The myosin coiled-coil is a truly elastic protein structure.</a> Nat. Mater. 1, 232-235 (2002).</li><li>Root, D. D., Yadavalli, V. M., Forbes, J. G., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coiled-coil+nanomechanics+and+uncoiling+and+unfolding+of+the+superhelix+and+alpha-helices+of+myosin.">Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin.</a> Biophysical Journal. 90, 2852-2866 (2006).</li><li>Nishizaka, T., Miyata, H., Yoshikawa, H., Ishiwata, S., Kinosita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbinding+force+of+a+single+motor+molecule+of+muscle+measured+using+optical+tweezers.">Unbinding force of a single motor molecule of muscle measured using optical tweezers.</a> Nature. 377, 251-254 (1995).</li><li>Gawalapu, R. K., Root, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+labeling+and+computational+analysis+of+the+strut+of+myosin's+50+kDa+cleft.">Fluorescence labeling and computational analysis of the strut of myosin's 50 kDa cleft.</a> Arch. Biochem. Biophys. 456, 102-111 (2006).</li><li>Kellermayer, M. S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+and+manipulating+individual+protein.">Visualizing and manipulating individual protein.</a> Molecules Physiol. Meas. 26, R119-R153 (2005).</li><li>Shimizu, T., Dennis, J. E., Masaki, T., Fischman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+arrangement+of+the+myosin+rod+in+vertebrate+thick+filaments:+immunoelectron+microscopy+with+a+monoclonal+antibody+to+light+meromyosin.">Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.</a> J. Cell Biol. 101, 1115-1123 (1985).</li><li>Godfrey, J. E., Harrington, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-association+in+the+myosin+system+at+high+ionic+strength.+I.+Sensitivity+of+the+interaction+to+pH+and+ionic+environment.">Self-association in the myosin system at high ionic strength. I. Sensitivity of the interaction to pH and ionic environment.</a> Biochemistry. 9, 886-893 (1970).</li><li>Root, D. D., Stewart, S., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+docking+of+myosin+and+actin+observed+with+resonance+energy+transfer.">Dynamic docking of myosin and actin observed with resonance energy transfer.</a> Biochemistry. 41, 1786-1794 (2002).</li><li>Xu, J., Root, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Selection+during+Weak+Binding+at+the+Actin+and+Myosin+Interface.">Conformational Selection during Weak Binding at the Actin and Myosin Interface.</a> Biophys. J. 79, 1498-1510 (2000).</li><li>Sattin, B. D., Pelling, A. E., Goh, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+base+pair+resolution+by+single+molecule+force+spectroscopy.">DNA base pair resolution by single molecule force spectroscopy.</a> Nucleic Acids Res. 32, 4876-4883 (2004).</li></ol>

When converting a movie to a digitally thresholded representation, it is crucial for the thresholded image to maintain the same area in each frame of video. Because the beads in a bead pair move independently of one another, any drift in the thresholded areas can also cause the relative distances between the centroids of the beads to drift and introduce significant error. Controlling the threshold area reduced the error five-fold in the distance measurements from 26 nm down to 5 nm. It is also crucial to obtain an acc...

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demonstrating the uses of the novel gravitational force spectrometer to stretch and measure fibrous proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

Watch this Scientific Journal Video about Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins at JoVE.com

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

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