The heavy chain is shown in blue, the essential light chain is shown in green, and the regulatory light chain is shown in red.
(Figure 1 Rayment et. al.)
Figure 2: Dimer Pairing Diagram
During muscle contraction, the myosin and actin filaments slide past each other and cause the muscle to shorten. When broken down, myosin filaments are made up of many myosin heads which bind to the actin and then rotate causing the myosin and actin filaments to slide past each other (Bagshaw 1993). Each myosin head is made up of three main helical protein strands; the regulatory light chain (RLC), essential light chain (ELC), and the heavy chain (shown in figure 1) . (Rayment et al. 1993). These myosin heads are situated in pairs, known as dimer pairs, and they are joined together at their base (shown in figure 2).
The heavy chain is shown in blue, the essential light chain is shown in green, and the regulatory light chain is shown in red.
(Figure 1 Rayment et. al.)
Figure 2: Dimer Pairing Diagram
Recent studies of muscle dynamics have been focused mainly upon the action of the two filaments, and of the myosin heads individually, not as a pair. From these studies, our knowledge of muscle action has become relatively complete, but the purpose of the pairing of the myosin heads is still unknown. Our current model for muscle contraction deals only with the individual myosin heads, so there may be a key step missing which involves the myosin heads as pairs.
Since the interactions of myosin head dimer pairs has been an overlooked factor in muscle contraction. Therefore the purpose of this research will be to study how the myosin head pairs interact with each other and with the actin filament. In order to detect the distance between the two heads in the pair, fluorescence energy transfer (FET) testing will be done.
FET is a relationship between two molecules where they exchange energy while in their excited states. When a pair of these molecules, one known as the acceptor probe and the other as the donor probe, are struck by light, the donor probe becomes excited. Then after a short period of time the probe releases this energy in the form of fluorescent light. The time from excitation to emission is known as the lifetime of the excited state. The presence of the acceptor probe shortens the lifetime of the donor probe. The closer the probes are, the shorter the lifetime, and the greater the distance between them, the greater the lifetime (Campbell and Dwek 1984). The probes are attached to the muscle fibers via the RLCs because it is the most reliable means for attachment.
The RLCs can be removed individually from the pair because the two RLCs in the pair possess a property known as negative cooperatvity. This means that one RLC has a much stronger bond with the heavy chain than the other so it can be removed from the myosin much more easily than the other RLC in the pair (Bagshaw 1993).
Theoretically the ratio of the masses of the RLC and ELC within each myosin head chains should be one-to-one. A urea gel separates the different parts of the muscle fiber into three different bands. The gel separates the bands using a charge and a web like solution. These bands can be analyzed to establish a ratio between the RLC and ELC. This ratio can be used to determine the accuracy of the RLC exchange.
From the results of these experiments, and those of a previous experiment (Baker et al. 1998), it will be possible to estimate with a high degree of certainty the formation of the heads will be in each physiological state (rigor, relaxation, and contraction). Rigor State is when neither ATP nor Ca2+ are present, and the myosin heads are bound to the actin. Relaxation State is when ATP is present, but Ca2+ is not, so the myosin heads are not bound to the actin. Contraction State is when both ATP and Ca2+ are present, and the myosin heads are bound to the actin.
The most likely result is that when the muscle changes from rigor to relaxation the distance between the myosin heads will increase and as the muscle changes from rigor to contraction the distance between the heads increase. The structural changes that are most likely to occur are shown in figure 3. The results of the experiments will be used to help gain a better understanding of how muscle works on a very specific level. The goal of this research is to obtain enough reliable and accurate data to provide a good solid base for future research.
The first two diagrams are of rigor, the next two are of relaxation and the last two are of contraction.
Part 1: Fiber Harvesting
The muscle fibers that were used for this experiment were taken from live scallops. The advantages to using scallop muscle are that it is striated similarly to human muscle and that the light chains can be exchanged easily (Vilbert 1992).
Part 2: Fiber Labeling
In order to prepare the muscle fibers for testing, the myosin heads of the extracted fibers had to be modified so that either a donor or acceptor probe molecule is present on the myosin heads in the muscle fiber.
The first step of the light chain exchange was to remove an RLC from one of the myosin heads of each pair in the fiber. To accomplish this, the magnesium bonds between the RLC and the heavy chain were broken. A solution known as EDTA was used to break this bond as the fibers were washed with EDTA for 30 minutes. Only one of the RLCs in the pair was removed during this extraction because the pairs possessed negative cooperativity. After the first extraction, then a labeled chicken gizzard RLC was added onto the heavy chain where the extracted RLC was originally. The RLC was labeled in another part of the lab. The chicken gizzard RLC was labeled with a donor molecule (Eosin). The magnesium bonds were reformed by washing the fibers with a solution containing magnesium. After the labeled RLC was added to the myosin heads, it had the stronger of the two bonds within the pair. The same extraction and readdition processes were used for the second exchange, except the chicken gizzard RLC was labeled with the acceptor probe (tetramethyl rhodamine iodoacetamide). Several fibers were saved after the first extractions for later gel testing.
Part 3: Exchange Effectiveness Assay
Once the light chain exchange was completed, then a urea gel was used to test the precision of the exchange, by establishing a ratio between the masses RLC and ELC. For native fibers and fibers with readded RLC, the ratio theoretically should have been one-to-one. For the fibers with extracted RLC the ratio should have been one-to-two. When the results of the gel were reliable and fairly accurate, the fibers were used for fluorescence testing. If the results were not accurate or reliable, then the exchange process was repeated using fresh fibers.
Part 4: Fluorescence Testing
First, the fiber was mounted on a stand (shown in figure 4) so that the FET could be measured, then a laser varied the light intensity over 20 different intensities, and the computer then calculated the average of all these lifetimes. During the testing, the fiber was immersed in solution that simulated one of the three physiological states: rigor, relaxation, and contraction. Each fiber was tested in each state until a consistent lifetime value had been established for each state, this value was known as t da for the respective stae. A fiber labeled with only donor probes was tested for the three different physiological states until a t d was established for each state. These two values were then plugged into this equation:
E=1-(t da/t d ) = (R06)/( R06+R6 )
Where E is the efficiency of the energy transfer, R is the distance between the probes, and R0 is a constant. After consistent t d and t da values have been established for contraction, then values for relaxation and rigor must be found.
Figure 4: A Diagram of the FET testing setup.
The majority of the data from the experiments is in the form of gel data. Fluorescence testing could not be done until the ratio of the RLC and ELC was within about a 10% to 20% deviation from the theoretical values. Early gel data is shown in figure 5. Here the values have a large deviation from the theoretical values. Data from testing later in the project is shown in figure 6. The values were significantly closer to the expected values than those from earlier in the project.
Raw lifetime data from the fluorescence energy transfer tests are displayed in figure 7. Distance data from the FET tests is displayed in figure 8.
The FET data from the experiments proved to be very unreliable. The majority of tested fibers showed either no energy transfer or no consistent distance change between the different states. The methods of the exchange process methods have been established to be accurate and reliable, but the data from the FET testing proved to be unreliable. There was no consistency among the FET tests as shown in graph 4, so there is no basis for making any conclusion regarding the change in the distance between the different states. Even fibers that showed they were capable of contracting while floating free in a solution, the results were still very unreliable. So this would suggest that it was the testing procedure, or possibly the effectiveness of the probe which caused the poor reliability. The majority of the lifetime data had values that were either too inconsistent or had too much error in their calculation to be used to calculate distance. The donor only lifetime value should remain constant in each of the different states because the probe is not being affected by any outside factors. The value of t d should remain constant in each of the different states, so the values in figure 8 suggest that there was no change in distance as the muscle changes states.
Also, the majority of the data is in the form of gel data because the extractions were not going well enough to use for FET testing. It was useless to do FET testing with fibers that showed poor exchanges, so the problems with the exchange process and gel testing had to be dealt with before FET testing could be done. Over the course of the experiment the numbers from the gels became consistently more accurate as the project progressed as shown in graphs 1 and 2. This suggests that had more time been available, more FET testing could have been done to conclusively determine the root of the problem. It was discovered that the most successful exchanges were done on fibers that were harvested and modified in the same day.
The effect of the light chain exchange process on the effectiveness of muscle contraction is unknown, so a way to test the function of the modified fiber needs to be added to the methods. With the readditions, The best readdition results came from RLC that had very recently been labeled with its respective probe. This is most likely because the bonds between the fibers and the RLCs act more predictably when the fibers are fresh. After the fibers had been extracted from the scallop, it was not guaranteed to behave as a living fiber. Living situations can only be simulated because the muscle had been removed from its native place, so there was no way to know for sure whether the fiber will still behave as a living fiber. This presents some very serious problems for testing which still need to be ironed out. Attaching the muscle fiber to a tensiometer during the fluorescence testing could very easily have done this. The tensiometer would show if the fiber was still capable of contracting. Because of equipment restrictions, testing on both contraction and distance between the heads could not be done.
Also, because there was an absence of energy transfer in a significant portion of the FET testing, there might have also been a problem with the probes. If the probes do not exchange energy as they are theoretically supposed to, then the data from the FET testing does not reveal anything. Testing with a different combinations of probes should be done to find a reliable pair.
The main conclusion that can be drawn from this experiment is that the methods for testing need to be modified. Either the equipment, the FET probes or the exchange process needs to be modified so that further successful research may be done. The exchange process was working well, but the FET testing was not, so final, conclusive data was not obtained. If this project were to be continued, the first step to attempt to alleviate the problems with FET should be to reevaluate the equipment set up.
I would like to thank all of the members of Dr. Thomas’s Lab at the University of Minnesota. I would especially like to thank Diane Eschliman, Dr. Dave Thomas, Lois Fruen, and Dr. Jacob Miller for all their help throughout this project.
Allen, T.S., N. Ling, M. Irving, and Y.E. Goldman. 1996. Orientation Changes in Myosin Regulatory Light Chains Following Photorelease of ATP in Skinned Muscle Fibers. Biophysical Journal. 70: 1847-1862.
Bagshaw, C.R. 1993. Muscle Contraction. London: Chapman & Hall, 1993.
Baker, J., I. Brust-Mascher, S. Ramachandran, L. LaConte, D.D. Thomas. 1998. A Large and Distinct Rotation of the Myosin Light Chain Domain Upon Muscle Contraction. Proc. Natl. Acad. Sci. USA. 95, 2944-2949
Brust-Mascher, I., L. LaConte, J. Baker, D.D. Thomas. Myosin Structure Changes Upon Muscle Activation but Not ATP Hydrolysis. Submitted to Biochemistry.
Cahntler, P.D. and T. Tao. 1986. Interhead Fluorescence Energy Transfer Between Probes Attached to Translationally Equivalent Sites on the Regulatory Light Chains of Scallop Myosin. J. Mol. Biol. 192:87-99.
Campbell, Iain D. and Raymond A. Dwek. Biological Spectroscopy. California: The Benjamin/Cummings Publishing Co., 1984.
Lymn, R.W. and E.W. Taylor. 1971. Mechanism of Adenosine Triphosphate Hydrolysis by Actomyosin. Biochemistry 10:4617-4623.
Rayment, I., H.M. Holden, M. Whittaker, C.B. Yohn, M. Lorenz, K.C. Holmes,
and R.A. Milligan. 1993. Structure of the Actin-myosin Complex and Its Implications for Muscle Contraction. Science 261:58-65.
Thomas, D.D., Ramachandran, S., Roopnarine, O., Hayden, D.W. and Ostap, E.M. 1995 The Mechanism of Force Generation in Myosin: A Disorder-to-Order Transistion, Coupled to Internal Structural Changes. Biophys. J. 68, 135s-141s.
Vibert, P. 1992. Helical Reconstruction of Frozen-Hydrated Scallop Myosin Fillaments. J. Mol. Biol. 223:661-671.
Xiao, Ming, T. Chakrabarty, R. Cooke, P.R. Selvin. 1999. The Structure of the Myosin Dimer: Measuring Light Chain to Light Chain Distances Using Lanthanide-based Resonance Energy Transfer. Biophys. J. 76: A34.