The Effect of Familial Hypertrophic Cardiomyopathy on the Structural Dynamics of Regulatory Light Chain Protein

Richard Timm
Breck School
Biochemistry

Background

Introduction

Methods

Results

Discussion

Bibliography

Background

Familial hypertrophic cardiomyopathy (FHC) is a heart disease that is caused by single amino acid mutations in a muscle protein called myosin. Having the disease results in an increase in the thickness of the left ventricular wall and ventricular septum of the heart, which can restrict blood flow. FHC is a frequent cause of sudden cardiac arrest in young athletes and affects about 0.2 percent of the population. This translates to roughly 500,000 people in the United States alone.

The major contractile proteins of cardiac muscle are myosin and actin. Myosin consists of three major proteins: one heavy chain and two light chains (LC). Myosin is one of the main contractile proteins of muscle and interacts with actin to cause muscle contraction. One LC is the regulatory light chain (RLC) and the other is the essential light chain (ELC) (Figure 1). Single amino acid mutations that cause FHC are found in all three sub-proteins of myosin. The focus of my work is to study the structural consequences of one of the RLC FHC mutations on muscle fibers.

Introduction

There are seven different mutations in regulatory light chain that can result in FHC (Poetter, 1996). Previous work has shown that the biochemical properties of FHC diseased myosin are impaired compared to wildtype myosin (Roopnarine and Leinwand, 1998), thus making it important to determine the relationship between the onset of the disease and the function of the protein. A possible reason that this disease causes problems is that it changes the structure and movement of the protein myosin (Roopnarine, 1998). I. Rayment proposed that the LC domain of myosin acts as lever arm that changes its angle to produce force during muscle contraction (Rayment, 1995). Therefore, it is important to determine how the FHC mutations perturb this structural function of the LC domain. Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for detecting structural changes, which are determined from the line shapes of the EPR spectrum of spin labels attached to cysteine residues on the protein. This mutation occurs on a flexible region of RLC. The hypothesis of this research is that FHC affects the structural dynamics of a specific region in RLC. The goal was to test this hypothesis using EPR spectroscopy to test RLC for structural changes.

The FHC RLC mutation that was studied in this experiment is the Proline94 substitution with arginine (Pro94Arg). This mutation occurs on a flexible region of RLC that links its two globular domains. However, because the wildtype RLC does not contain a cysteine residue, which is necessary for attaching the spin label, a cysteine mutation was created at Ala92 in close proximity to the FHC mutation. This mutant RLC (Ala92Cys) was then used as the background for introducing the FHC mutation Pro94Arg (Figure 2).


Figure 2: Locations of cysteine (green) and FHC (yellow) mutations on RLC

In these studies, two separate cultures of E. coli were used to over-express wildtype and mutant RLCs, and the proteins were purified by an anion exchange DE-52 column. The Ala92Cys and Pro94Arg mutants were spin labeled with 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (FDNA) then exchanged in to rabbit muscle (Figure 3). Dr. Roopnarine handled all of the rabbit tissue herself since I did not want to encounter problems. Then preliminary EPR tests were run on the Ala92Cys mutant to use as a background for the Pro94Arg mutant. These results and further work are being presented at the Biophysical Society meeting in New Orleans during February 2000 (Roopnarine, Timm, Meland).


Figure 3a: Location of FDNA label on RLC

Figure 3b: Close-up of FDNA label


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Methods

 Mutagenesis of RLC. DNA primers containing the cysteine mutation were created in the Microchemical DNA Sequencing Facility at the University of Minnesota for use in mutagenesis. Using the "QuikChange Mutagenesis Kit," new plasmids containing the mutations were created by a polymerase chain reaction. These were then introduced into E. coli, which were used for expression of RLC. The expression plasmids were uniquely designed to translate the foreign protein. After growth of the E. coli, a single colony was isolated and the DNA was purified. The DNA was sent to a sequencing facility to determine if the amino acid mutation was present in the RLC gene. The purified plasmid containing the RLC with the mutation was then transformed into a cell line that would allow expression of the RLC, JM109DE3.

Expression and purification of RLC. Then the E. coli containing the mutant RLC was used to inoculate several small cultures, which were inoculated with ampicillan to kill bacteria not containing the RLC gene. A 15% Tris-HCl gel was run on the cultures so that the sample that had over-expressed the RLC the most could be cultivated. The E. coli bacteria were grown in four 2-Liter flasks. Isopropylthiogalactoside was added to induce expression of RLC on a larger scale.

The E. coli were subjected to a fatal freeze-thaw, then the cell membranes were opened with lysozyme. The DNA was digested using DNase. The RLC was protected in insoluble inclusion bodies, which were spun in a centrifuge to de-suspend them. The supernatant was then saved for gel analysis.

The insoluble inclusion bodies were incubated in highly concentrated urea, releasing the RLC into solution. To further purify the RLC, the urea containing dissolved RLC was run through an anion exchange column. In the column, the negatively charged RLC bonded to the positively charged column. A gradually increasing gradient of NaCl was then run through the column, forcing the protein out. The relative amount of protein that came out of the column was determined by the absorbance at 280 nm of light as the solution flowed out of the column and into test tubes. After the solution containing protein flowed out of the column, the test tubes that showed high amounts of protein were taken for gel analysis.

The RLC samples were dialyzed to remove urea. To dialyze the solution, it was poured into semi-permeable dialysis tubing. The tubing held the protein while the urea solution was highly diluted in an ammonium bicarbonate solution. The protein in ammonium bicarbonate was then put in a round bottom flask and lyophilized. Lyophilization is a process where the solution is frozen and then sublimates in a strong vacuum, leaving pure RLC as residue on the bottom of the flask. The volatile salt ammonium bicarbonate vaporized following the equation:

NH4HCO3 (s) ==> NH3(g) + CO2(g) + H2O(g)
The purity of the RLC was determined by sodium dodecyl sulfate polyacrylemide gel electrophoresis (SDS PAGE) analysis using a 15% Tris-HCl gel (Figure 4).

Spin labeling the RLC. The RLC was dialyzed extensively against NaPi buffer, and the FDNA spin label and RLC were allowed to react. Un-reacted FDNA was removed through centrifugation.

Exchange of RLC into muscle fibers. Dr. Roopnarine, my supervisor, performed this portion of the procedure, since it involves the handling of animal tissue. Rabbit muscle fiber bundles stripped of RLC were incubated with FDNA labeled RLC, which bonded spontaneously to each other. The fibers were put into 25mL glass capillaries and washed with NaN3 and a 15% SDS PAGE was used to determine the RLC content of the fibers. The fibers were used immediately for EPR experimentation.

EPR Spectroscopy. Conventional EPR spectra were acquired with a model ESP 300 spectrometer. EPR spectra of oriented muscle fibers were acquired with a TM110 cavity modified to hold a capillary parallel to the magnetic field.

Analysis of Spectroscopic Data. Each spectrum had to be baseline-corrected and normalized to unit-spin concentration. The rotational correlation time (treff) was found using the following equation, where 2TŐ|| and 2T|| are the splitting between the outer extrema of the experimental spectrum and a rigid limit spectrum, respectively.

The rigid limit spectrum (2T|| = 64 " 0.1 G) was obtained from labeled RLC immobilized on muscle fibers. The constants, a and b are defined a = 5.4 * 10-10 and b = -1.36.
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Results

 SDS PAGE analysis of the fractions that eluted during washing the column showed that the all the RLC bound to the column. Further analysis of the fractions that eluted with the linear gradient of NaCl showed the RLC was successfully purified by anion exchange chromatography. Figure 4 shows purification of proteins heavier and lighter in molecular weight as compared to RLC. The yield of purified protein was found to be approximately 50 mg per liter culture used to grow E. coli.

The Cys92RLC and Cy92Pro94ArgRlc were also successfully spin-labeled with the FDNA SL. Analysis of the peak widths for Cys92RLC splitting between extreme outer peaks, (Figure 5) (2TŐ|| = 55 " 0.1 Gauss) and determination of the effective rotational correlation time resulted in a t = 7 ± 0.3 ns, which is typical of a protein tumbling in solution with the molecular size of RLC (~ 20 kD). Analysis of the spectrum for Cys92Pr094ArgRLC gave similar results.
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Discussion

Two mutant RLCs were created using a Mutagenesis kit and a polymerase chain reaction (PCR). Ala92Cys was the first mutation to be created in the RLC, which was then used as a background for the FHC mutation (Pro94Arg). So the RLC with the FHC mutation contained both the Ala92Cys mutation and the Pro94Arg mutation. Isolation and sequencing of both of these plasmids showed no further mutations had been introduced as a result of the PCR experiment. After the plasmids containing the cysteine mutation and the plasmids containing the FHC and cysteine mutations had been created, they were introduced into E. coli and the RLC was successfully purified. Successful purification means that future work to study effect of FHC mutation on structure and dynamics is possible.

The spin labeled Cys92-RLC and the Cys92Pro94Arg-RLC had similar EPR spectra typical of small proteins tumbling in solution. The calculated effective rotation correlation times of both labeled RLC were very similar, ~ 7 nano-seconds each.

The use of the rat ventricular RLC means that this work can be compared with other animal studies such as transgenic mice (where the FHC is created in the mouse and the effects of the FHC mutations are observed in vivo).

It is predicted that the presence of the Pro94Arg mutation in the linker region of the RLC may perturb the dynamics that this region may be involved in during contraction. Future studies of the labeled RLC in muscle fibers will determine this.
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Bibliography

1. Rayment, I., Holden, H. M., Sellers, J. R., Fananapazir, L, and Epstein, N.D. 1995. Structural interpretation of the mutations in b-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy. Proc. Natl. Acad. Sci. U.S.A. 92:3864-3868.

2. Poetter, K., Jiang, H., Hassanzadeh, S., Master, S.R., Chang, A., Dalakas, M.C., Rayment, I, Sellers, J.R., Fananapazir, L., and N.D. Epstein. 1996. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nature Genetics 13: 63-69.

3. Roopnarine, O., and Leinwand, L.A. 1998. Functional analysis of myosin mutations that cause familial hypertrophic cardiomyopathy. Biophys. J 75:3023-3030.

Acknowledgements

I would like to thank Dr. Roopnarince, Dr. Thomas, Dr. Miller, and everyone else at Thomas Lab for their guidance in my research.
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