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

Summary

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

Abstract

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 evidence^{2, 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.

Protocol

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 dynamically oriented in relation to the vector so the force of gravity can impart piconewton-range force loads to the samples. The camera replaces the....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-Aminopropyltriethoxysilane	Polysciences, Inc.	919-30-2
Acetone	Fisher Scientific	A18P-4
Pyridine	Sigma-Aldrich	110-86-1
Glutaraldehyde	Fisher Scientific	G7776
Glycine	Research Organics	BP381-1
Tris	Sigma-Aldrich	9682T
Sodium azide	Amresco	71289
BSA	Sigma-Aldrich	AMR-0332-100G
NaCl	Sigma-Aldrich	S7653
EDTA	MSI	E9884
Nitrocellulose	Sigma-Aldrich	60443
N-N Dimethyl Formamide	Extracted from Large New	D4254
Rabbit skeletal myosin II	Zealand White Rabbits (7-8)	NA
MF30 antibody (9-10)	Developmental Studies Hybridoma Bank	MF30
MF20 antibody (6)	Hybridoma Bank	MF20
Lab microscope	Boreal	WW57905M00
Equatorial mount	Celestron	CG-5
Digital video cam	Sony Corporation	XCDV60
Caliper release	Cabelas	IA-415482
Compression spring	Jones Spring Co.	723
Extension spring	Jones Spring Co.	770
ImageJ	National Institutes of Health	NA
Fire-i drivers & application	Unibrain	3.80
Excel	Microsoft	NA