X-ray diffraction of muscle fibres

As a way of introduction, here is a representation of 2 sarcomeres from skeletal muscle, shortening according to the sliding filament hypothesis
An introduction to the mechanism of muscle contraction can be reached by clicking here or on the image above

Another project is described here: Measurement of Pi release in contracting muscle fibres

Time resolved small angle x-ray diffraction of single muscle fibres

A small part of the work carried out by this unit, but the part I am involved in, is x-ray diffraction experiments on single muscle fibres carried out at the Synchrotron Radiation Source in Daresbury, Cheshire.

Basics

When x-rays travel through a muscle cell, most of the x-rays travel undisturbed. A fraction however will be absorbed by the protein, or by the water of the cell or of the solution in which it is bathed. Some more will be scattered by the object. The scatter appears to be pretty random, but when the scattering molecules are arranged in a regular fashion, with repeating features arranged with spatial periodicities not too dissimilar in magnitude from the wavelength of the x-ray radiation, interference phenomena will cause some of the x-rays to be scattered discretely, causing spots to appear on a photographic film placed down-stream from the muscle fibre. The position, shape and brightness of the spots provide information about the disposition of the regular scattering features in the muscle cell. The experiments which we carry out are possible because of this quasi-crystalline nature of muscle cells. Furthermore, the scattering characteristics of the muscle change when the muscle's physiological state changes, i.e. when it is developing force or is shortening. This means that the brightness, shape and position of some of the spots depend on the muscle state. Our work aims at measuring, and in interpreting these changes, in order to understand the precise relationship between generation of force, breakdown of chemical energy (ATP hydrolysis) and changes in protein structure. How is the force generated, and how do the structural changes associated with force generation modify the kinetics of ATP hydrolysis?
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Teamwork

The experiments involve a number of collaborations with people from several laboratories. Below is a picture of Ronnie Burns, Ph.D. student, setting up the instrumentation on beam line 16.1. Sergey Bershitsky is nursing a muscle fibre whilst Andrey Tsaturyan is searching for an interpretation of our results. The only one sitting down is me. Sergey, from Yekaterinburg, and Andrey, from Moscow, collaborate in our experiments thanks to a grant from INTAS. Click on pictures to see larger versions (March 1996).

A diagram of the instrumentation we use is shown here. In this collaboration we study the changes in the diffraction pattern from permeabilised muscle cells undergoing rapid perturbations such as changes in length (length steps), changes in temperature (T-jump) or changes in substrate concentration (laser-pulse photolysis of caged-ATP). The analysis of the x-ray data is made possible thanks to Geof Mant, to the CCP13, a Collaborative Computing Project for the development of software for the analysis of fibre diffraction data, sponsored by the EPSRC and thanks to Liz Towns-Andrews , Station Manager at the SRS who constructed beam line 16.1 and makes our experiments possible.

A collaboration with Drs. Lombardi, Piazzesi (University of Florence), Irving and Dobbie (The Randall Institute, King's College London) investigates the structure/function relationship using intact muscle fibres. This work involves the measurement of changes in the cross-bridge orientation during mechanical perturbation of the contracting fibres. Here we observe changes on the time scale of 10 microsecond. The instrument used is illustrated here. Click on icons to see the set-up:

Here are pictures of the team members:
They are (from the left) Malcolm Irving & Vincenzo Lombardi setting up a muscle fibre, Ian Dobbie, at the keyboards, Malcolm in front of the wire chamber detector (with appropriate T-shirt), Vincenzo setting up the striation follower, Gabriella Piazzesi, dissecting, Massimo Reconditi also dissecting.
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More about our experiments

The last picture is a low angle diffraction pattern from a relaxed frog muscle fibre obtained at the synchrotron.The x-rays scattered by repeating features through the thickness of the muscle cell are scattered in a plane perpendicular to the muscle fibre. On the x-ray detector, this plane appears as a line, with bright spots indicating the repeating distances between the longitudinally arranged protein filaments. This line is called the equator. The x-rays scattered by features which repeat along the length of the muscle fibre (along the length of the filaments) are scattered in a plane called the meridian. The equator (horizontal) was attenuated electronically by 20 to show the equatorial reflections with the same intensity as the main meridional reflections. The pattern is averaged by symmetry, and was obtained from several fibres. However, this picture is a static picture.
The picture on the right shows how the pattern changes when the physiological state of a muscle fibre changes from relaxed, to active and to rigor. ( The equator is vertical, and electronically attenuated 20 times. ) The pattern provides information about the disposition of the muscle proteins responsible for the transformation of chemical energy into movement and mechanical work. The real interest of our work is to see how this pattern changes with time as the muscle proteins move during muscle contraction and relaxation. For this, we need to do time-resolved studies. The proteins move fast, so we need fast measurements, and a lot of x-ray photons to achieve this. Hence the need for a synchrotron, the most powerful source of x-rays to-date.
Currently, our time resolution is of the order of 0.1 ms. Not only do we need bright x-ray sources, we also need x-ray detectors which allow the observation and measurement of the diffracted photons. The speed of the detector, namely the numbers of photons it can count every second, is a determining factor. We use detectors developed and constructed at the Daresbury Laboratory. These are electronic detectors referred to as gas-filled wire chamber detectors. They resolve a 20cm square area, with a resolution of about 1000x1000 pixels and a maximum counting speed of 1 MHz. The counting efficiency is about 70-80% and the dynamic range is huge. These detectors suffer from a parallax problem which make them less than ideal for some protein crystallography applications, but they are state-of-the-art for low-angle diffraction studies. Have a look at my home page for more information about muscle contraction. Perhaps more powerful x-ray beams will become available in the UK in the future, but they are expensive. The European Synchrotron Radiation Facility in Grenoble is an example of a new generation of x-ray sources.
As an example, results obtained on beam line 16.1 in March 1996 with Sergey Bershitsky, Andrey Tsaturyan and Ronnie Burns can be seen by clicking here. The time course of tension and that of intensity changes of three x-ray reflections is shown, following the photolytic release of ATP from caged-ATP. The two main equatorial reflections (1,0) and (1,1), and the main meridional reflection change their intensity as the muscle fibre develops tension. The structural changes are fast, and appear to precede the tension change. The data are still noisy, and further experiments are planned to ascertain the time-courses. Comments or suggestions gladly received at m.ferenczi**ic.ac.uk
More info on x-ray diffraction and the Daresbury synchrotron. The analysis of x-ray data relies heavily on the programs XOTOKO and BSL developped at the SRS. A list of commands is shown here. New software is developped by CCP13.

Written by Ian Dobbie and Mike Ferenczi
m.ferenczi**imperial.ac.uk
Main modification dates: September 1994, November 1995, April 1996, Photos taken with a Kodak DC-50 added since February 1996

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