The purpose of this study was to engineer a single cysteine at position 72 on the myosin regulatory light chain (RLC) of smooth muscle. A cysteine has never been successfully engineered at position 72 on the myosin regulatory light chain. The naturally occuring amino acid at position 72 is methionine (M72). By engineering a cysteine in place of the methionine at position 72 and consequently producing the M72 mutant (M72C), the sulfhydryl group of the cysteine can function as an attachment site for a spectroscopic probe. This spectroscopic probe enables electron paramagnetic resonance (EPR) experiments on the interaction of myosin and actin in skeletal muscle contraction.

Hypothesis:

Position 72 will successfully produce a M72C mutant through site-directed mutagenesis.

There is much known about the mechanism of muscle contraction and the sliding-filament model; however there are still many uncertainties regarding the details of this process. A form of electron paramagnetic resonance (EPR) spectroscopy, which employs a nitroxide spin label that attaches to the sulfhydryl group of a cysteine, is used to detect orientational changes of myosin (see Fig. 1). EPR has been found particularly useful in gathering information regarding the interaction between myosin and actin because it is both site specific and sensitive to rotational motion.

The regulatory light chain of the myosin protein of a chicken gizzard smooth muscle has one naturally occurring cysteine and that cysteine is found at position 108. In EPR, a nitroxide spin-label can attach to the sulfhydryl group that only occurs in the cysteine amino acid. The naturally occuring amino acid at position 72 is methionine. In order to allow a nitroxide spin-label to be attached at this site, it is necessary to engineer a cysteine in place of the methionine through site-directed mutagenesis. In order to ensure that the nitroxide spin-label does not attach to position 108, the cysteine at this site must be replaced with a different amino acid. Alanine is usually chosen to replace the cysteine because it is of similar size and therefore is a conservative change (Smith, 1998).

Successful EPR has been performed at position 108 on the RLC of myosin. Thus far, EPR has not been performed on any other site on the regulatory light chain of myosin.

Two cultures of JM109 competent cells containing recombinant phagemid DNA were prepared by harvesting two individual tetracycline resistant colonies from a fresh plate and placing each in 5 mL of culture medium containing 2.5 mL tetracycline. The cultures were shaken overnight at 37°C. Two new cultures containing 25 mL of culture medium, 500 mL of the overnight cultures, and 62.5 mL of tetracycline were shaken at 37°C for 30 minutes. To the cultures, 200 mL of R408 were added. The cultures were then shaken at 37°C for another six hours. The cultures were put into centrifuge tubes and spun in the GSA rotor at 8600 rpm for 15 minutes. The supernatants were transferred to new tubes and spun for another 15 minutes. To each resulting supernatant, 7 mL of phage precipitation solution were added, and the solutions were kept at 4°C overnight. The solutions were spun in the SS-34 rotor for 15 minutes at 8600 rpm. The supernatants were drained and 400 mL of TE buffer were added to the remaining pellets. Each pellet was removed from the side of the tube by swirling a pipette tip inside the tube. The solutions were transferred to microcentrifuge tubes. In order to lyse the phage, 400 mL of chloroform:isoamyl alcohol (24:1 by volume) were added to the solutions. The solutions were vortexed for one minute and spun at 12000 x g in a microcentrifuge for five minutes. The upper aqueous phase (containing phagemid DNA) of each solution was transferred to a fresh tube. Identical extractions were then performed three times with a phenol:chloroform:isoamyl alcohol (25:24:1 by volume) mixture and once more with chloroform. To the solutions, 150 mL of 7.5M ammonium acetate and 900 mL of cold 100% ethanol were added. The solutions were then kept at –20°C for 30 minutes. The solutions were spun in the microcentrifuge for 15 minutes at 12,000 x g and the pellets were aspirated. To each solution, 400 mL of cold 70% ethanol were added. Solutions were spun again and the pellets were once again aspirated.

An alkaline denaturation solution was prepared by adding 5.3 mL of PAlter-Ex1 double-stranded DNA template, 2 mL of 2M NaOH, and 2mM EDTA, to 12.7 mL of sterile H₂0. The solution was incubated at room temperature for five minutes. To the reaction, 2 mL of 2M ammonium acetate at pH 4.6 and 75 mL of 100% ethanol were added. The reaction was cooled at -70°C for 30 minutes, and the DNA was precipitated by centrifugation at top speed for 15 minutes. The supernatant was aspirated, and the pellet was washed with 200 mL of 70% ethanol. The reaction was centrifuged again, and the pellet was dried. The pellet was dissolved in 100 mL of TE buffer.

A solution was prepared containing 8.91 mL of the diluted M72C oligo, 5 mL of 5xT4 ligase, 0.5 mL of T4 polykinese, and 10.59 mL of H₂O. The reaction was incubated at 37°C for 30 minutes and then incubated at 70°C for ten minutes and kept at -20°C.

A mutagenesis solution was prepared containing 10 mL of phagemid single-stranded DNA, 1 mL of Ampicillin Repair Oligo, 1 mL of Tetracycline Knockout Oligo, 1.32 mL of M72C Mutagenic Oligo, 2 mL of Annealing

10X Buffer and 4.7 mL sterile H₂O. A control solution was prepared as well, containing 10 mL of double-stranded DNA (dsDNA), 1 mL of Ampicillin Repair Oligo, 5 mL of Tetracycline Knockout Oligo, 2 mL of Annealing 10x Buffer, and

2 mL of sterile H₂0. Both solutions were placed in a water bath at 75°C for five minutes. The blocks were placed at room temperature until the solutions reached 45°C and then placed on ice to 10°C.

Sterile 17x100mm polypropylene culture tubes were pre-chilled on ice. Frozen ES1301 mutS Competent Cells were removed from -70°C and were placed on ice until just thawed. The cells were gently mixed, and 100 mL of the cells were transferred to each culture tube. To 100 mL of the mutS cells, 1.5 mL of each mutagenesis reaction were added. The mutagenesis reactions were mixed with the cells by swirling the pipette tip while dispensing. The tubes were placed on ice for ten minutes. The cells were heat-shocked for 45-50 seconds in a water bath at 42°C and then placed on ice for 2 minutes. To each reaction,

900 mL of LB without antibiotic at room temperature were added. The reactions were shaken for 30 minutes at 37°C. To 4.5 mL of LB and 125 mg/mL of ampicillin, 500 mL of each transformation were added. The transformations were incubated overnight at 37°C with shaking.

Into a microcentrifuge tube, 1.5 mL of the overnight culture were transferred and spun at 12,000 x g for one minute. The supernatant was aspirated, and the bacterial pellet was resuspended in 100 mL of ice-cold

buffer I solution. The solution was incubated for five minutes at room temperature. To the solution, 200 mL of freshly-prepared buffer II solution were added in order to lyse the cells. The cells were mixed gently by inversion and were incubated on ice for five minutes. To the cells, 150 mL of buffer III solution were added and incubated on ice for five minutes. The cells were spun in the microcentrifuge at 12000 x g for five minutes. The supernatant was transferred to a fresh tube. Extractions using one volume phenol:chloroform:isoamyl alcohol (25:24:1 by volume) and one volume chloroform:isoamyl alcohol (24:1 by volume) were performed on the supernatant. To the supernatant, 2.5 volumes of 100% ethanol were added and the solution was mixed thoroughly and was allowed to precipitate 20 minutes at -20°C. The solution was centrifuged at 12000 x g for five minutes and the supernatant was aspirated. The pellet was rinsed with 1 mL cold 70% ethanol and dissolved in 49 mL of TE buffer and 1 mL of 10 mg/ml RNase A.

Sterile 17x100mm polypropylene culture tubes were pre-chilled on ice. Frozen JM109 competent cells were removed from -70°C and placed on ice until just thawed. The cells were gently mixed by flicking the tube, and 100 mL of the cells were transferred to each culture tube. To 100 mL of the competent cells, 1.5 mL of each plasmid DNA sample was added. The plasmid DNA samples were mixed with the cells by swirling the pipette tip while dispensing and were placed on ice for 30 minutes. The cells were heat-shocked for 45-50 seconds in a water bath at 42°C. The tubes were placed on ice for two minutes. To each sample, 900 mL of LB without antibiotic at room temperature were added. The samples were incubated for 60 minutes at 37°C with shaking. On an LB plate made with ampicillin and an LB plate made with tetracycline, 100 mL of each transformation were plated. The plates were incubated overnight at 37°C.

Following the first preparation of single-stranded DNA and the ensuing execution of the phosphorylation of oligonucleotides, mutagenesis, transformation of ES1301 mutS competent cells and plasmid prep, the results of a gel indicated that the DNA had been lost at some point in the process. A subsequent gel that tested the product of the single-stranded DNA preparation showed that there had never been any single-stranded DNA. The single-stranded DNA procedure was repeated, and a gel revealed that this second attempt was successful.

The freshly prepared DNA was used in the mutagenesis, transformation of ES1301 mutS cells, and plasmid prep procedures. A gel showed that at least one out of the last three procedures performed had been unsuccessful. From the overnight culture produced by the most recent transformation of ES1301 cells, 50 mL was transferred into 5 mL of LB with 125 mg/mL of ampicillin. After the new solution was shaken overnight at 37°C, a plasmid prep was performed. A gel suggested that there was a small amount of DNA; however, there was probably not enough to survive the following transformation into JM109.

Double-stranded DNA was prepared for use in a control for the mutagenesis procedure. Single-stranded DNA that would allow for ten mutagenesis procedures was also made. A gel indicated that these preparations had been properly performed. Mutagenesis was performed on the oligos M72C and A108C as well as two oligos that had been shown to mutate properly: M60C and A105C. A control was also used in the mutagenesis procedure.

After a plasmid prep and transformation into ES1301 cells had been performed on the mutagenic reactions, a gel indicated that there was very little DNA. This result suggested that some aspect of the mutagenesis procedure had failed. Since the mutagenesis procedure had been performed as carefully as possible, it was likely that one or more of the materials used in the procedure had caused a problem. Therefore, the procedure was done the same way, except fresh ampicillin was used. A gel showed that there was DNA.

Using the product of the last mutagenesis, the transformation into ES1301 cells, the plasmid prep, and the transformation into JM109 cells were performed. The resulting plates suggested that the transformation had been successful. There were very little colonies on the tetracycline plates and the ampicillin plates had many colonies. The mutated oligos were DNA sequenced mechanically. The results showed that the oligos had not mutated properly.

Upon request of the author, a fellow research student fabricated a computer model of a hydroxide spin label at the mutated position 72. The results indicated that position 72 might not be a good site to pursue.

Conclusion:

The results obtained through the automated DNA sequencing, as well as the results throughout this study, suggest that there was most likely a problem with the mutagenic oligo. Because the colonies were able to grow on the ampicillin plates and unable to grow on the tetracycline plates, it is clear that the antiobiotic resistance switched as is anticipated in the mutagenesis procedure. Therefore, it is unlikely that there was a something wrong with the procedure; rather, there was probably a problem with the oligo.

There are a few possibilities as to what may have been wrong with the mutagenic oligo in this study. First, it is conceivable that the oligo may have been defective. It is possible, although not very probable, that the company from which it was purchased may have incorrectly sequenced the oligo. Because the oligo was not sequenced after arriving at the laboratory, it was not determined whether the oligo was correct. It is more likely that the oligo did not anneal properly during the mutagenesis procedure. As a result of not annealing correctly, the oligo would not acquire a cysteine, yet the colonies that would grow in the overnight cultures would be ampicillin resistant.

The continued study of site-directed mutagenesis on myosin is important as it is integral to the further discoveries regarding the mechanism of muscle contaction. As scientists learn more about the role of myosin in the muscle and the structure of the myosin protein, important discoveries pertaining to muscle malfunction and possible treatments can be obtained.

In a muscle contraction, actin filaments slide past myosin filaments; however, the contraction is only possible in the presence of ATP. ATP binds to myosin and is quickly hydrolyzed, creating a myosin-ADP-P_i complex. The myosin heads can then attach to binding sites along the actin filament. When the myosin and actin filaments are bound together, the P_i is released and the subsequent dissociation of ADP causes the myosin heads to powerfully pull back the actin filament towards the center of the myosin filament. Following this power stroke, the ADP is released, and the myosin head detaches from the actin binding site. The myosin head is then capable of binding to ATP and accordingly attaches to a new binding site along the actin filament. Ultimately, this ATP-ADP cycle causes the myosin and actin filaments to overlap. Because neither filament changes length in the process, the sarcomere, which contains the protein filaments and is the smallest until of contraction, shortens. As many sarcomeres shorten, the muscle contracts (Stryer, 1995).

Baker, J.E., I. Brustmascher, S. Ramachandran, L.E.W. LaConte, and D.D. Thomas. 1998. A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction. Proceedings of the National Academy of Science, 95:2944 2949.

Holmes, K.C. 1997. The swinging lever-arm hypothesis of muscle contraction.Current Biology, 7: R112-R118.

Goldman, Y. 1998. Wag the tail: structural dynamics of actomyosin. Cell. 93: 1- 4.

Meland, William. "Virtual Scanning Mutagenesis of Myosin for Spectroscopic Studies of Muscle." Diss. U of Minnesota, 1998.

Smith, Wendy. Personal Interview. 18 May- 20 August. 1998.

Stryer, Lubert. Biochemistry. 4^th ed. New York: W.H. Freeman, 1995.

Thomas, D.D., S. Ramachandran, O. Roopnarine, D.W. Hayden, and E.M. Ostap. 1995. The mechanism of force generation in myosin: a disorder-to-order transition, coupled to internal structural changes. Biophys. J. 68: 135s-141s.

Thomas, David. Personal Interview. 18 May- 20 August. 1998.