Student Research at Duke

Running the PCR

Agarose Gel

I spent the whole day yesterday running PCR and gels. As I mentioned previously, I didn’t have a successful clone so I started over. The PCR band for the overlapping fragment turned out wonderful! And my next step would need to CAREFULLY purify the DNA by cutting the bands. I’ll have to think of a way to optimize my yield for the overlapping DNA fragments.

Kinesins are motor proteins that “walk” on the microtubules by utilizing the energy derived from ATP hydrolysis. However, it has been quite controversial in determining which step of the ATPase activities is responsible for the power stroke, or the walking step, for the motor proteins. There are two mainstream models proposed to explain the possible mechanisms for motor protein power stroke. The first model claims that it is the step of ADP release that triggers the power stroke of kinesin protein. The alternative model proposes that it is upon ATP binding that the power stroke occurs. The following three papers gave me great insight into each of the model, allowing me to make side-by-side comparison on evidences supporting each model.

1. A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd Nicholas F. Endres1, Craig Yoshioka2, Ronald A. Milligan2 and Ronald D. Vale1

2. Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd
The EMBO Journal (2003) 22, 5382–5389 Mikyung Yun1, C.Eric Bronner2, Cheon-Gil Park1, Sun-Shin Cha3, Hee-Won Park1 and Sharyn A. Endow2

3. Kinesin motors as molecular machines Sharyn A. Endow BioEssays 25:1212-1219, 2003.

The information I learned here is crucial for my project this summer, which involves creating an Ncd mutant by mutating the glycine residue at the neck region. In order to predict the behaviors of the mutant, a basic understanding of how motor proteins give power stroke is critical. After studying through tremendous amount of information included in these papers, let me try to explain both models based on my understanding.

It is important to recognize that these two models are not entirely different. The common ground for both models lies on the basic structure of the kinesin proteins.
ATP hydrolysis introduces “strain” in the kinesin proteins, altering the protein conformation from its original position. The release of the stored strain creates a power stroke to bring the altered conformation back to its original state. In order to accomplish the power stroke, four basic components are needed: a spring, a lever, a hinge, and a latch. A spring is the elastic component that store and release strains. The hinge enables the lever, or coiled coil stalk region, to amplify the conformational change. And the latch at the nucleotide binding site determines the nucleotide states, whether is ATP binding state, ADP binding state, or nucleotide-free state.

Model ONE: Kinesin proteins produce power stroke upon ADP release.
For mutant Ncd N600K, one head (motor domain H1) and the stalk are found to rotate together while the other head (H2) does not involve in the rotation. In the other words, two heads appear to be asymmetric relative to the coiled-coil stalk. Interestingly, the nucleotide state for each of the motor domain (H1 and H2) differs: H2 only loosely associates with ADP while H1 is stably bounded to ADP.

So what makes the stalk rotate with H1? N600K interacts with the R552 of H2, and as a consequence, the neck is not able to interact with the motor region (H2). This allows the rotation of the stalk. The interaction of N600K and R552 also results in the dissociation of ADP from the binding site on H2, which explains the different nucleotide binding states. The new crystal structure of this particular mutant involves two heads with different nucleotide binding states, and the loosely attached ADP resembles the release of ADP that might bring forth the power stroke.

Model TWO: Kinesin proteins produce power stroke upon ATP binding. When Ncd binds to the microtubule with single head, ADP is released. After the hydrolysis products are released, Ncd is able to bind to a new ATP. The ATP binding promotes the rotation of the stalk. The following protein isomerization process results in the unbinding of Ncd from the microtubule.

My next step would be trying to simulate how my mutant would turn out according to these two models.

Even though our lab studies the kinesin motor protein which walks on the microtubule filament, learning more about the actin filament assembly wouldn’t hurt! I just read an interesting paper that talks about how Arp2/3 protein induces actin polymerization.

Through the polymerization of actin filament tail attached to its surface, Listeria monocytogenes, a type of mobile bacterium, moves around in the infected cells. According to this study, Arp2/3 protein complex is a crucial element that is responsible for nucleating the actin filament attached to the Listeria surface. Through column chromatography, researchers were able to purify an eight-polypeptide complex that would induce the polymerization of actin filament. Three kinds of columns were run in the following sequential manner in order to obtain this purified complex: Q-sepharose column (Anion chromatography), S-sepharose column (Cation chromatography), and Superose-6 gel filtration chromatography. A model is proposed that ActA on the bacteria surface activates the protein complex, enabling the complex to serve as a nucleating site for the elongation of actin filament. Once the nucleating process is initiated, the complex may dissociate from ActA and become part of the tail attached to the Listeria surface.

Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes by Matthew D. Welch, Akihiro Iwamatsu & Timothy J. Mitchison

Me and my co-worker, Mark

The following is a short summary about what I have done so far. I have been trying to create the Ncd G347D mutant. If all the steps had gone successfully, I should have gotten my mutant by the end of today. Due to some technical difficulties, my colonies didn’t contain the plasmid of interest, and thus not being able to obtain the mutant DNA. As I mentioned in my previous post, my job involves mutating the glycine 347 to an aspartic acid. To create this mutant from scratch, I used a method called the “site-directed mutagenesis by overlap extension using the polymerase chain reaction.”

(Steffan N. Ho^a, Henry D. Hunt^a, Robert M. Horton^b, Jeffrey K. Pullen^a and Larry R. Pease. Gene, Vol. 77, 15 April 1989, Pages 51-59.)

This is a commonly used molecular method in order to introduce point mutation into the wild type gene. Dr. Endow ordered me primers that were required to carry out this method. After each PCR reaction I had to run an agarose gel in order to verify that I have gotten the correct fragments. Running the gels are check points that are extremely crucial for the success of overall experiment. Ever since I started to create this mutant, I had run for over 20 gels, and I only become more and more efficient at doing this particular task. Once get the mutated gene fragments (although it’s hard to be confident that I had introduced the mutation for sure), I ligased the fragment with the vector DNA, forming a plasmid. Then the next step is to carry out a transformation by plating the competent cells and the plasmid onto the media plate with antibiotics. I have gotten very few colonies that were likely to contain the plasmids. Even so, I still carried out mini preps after harvesting the culture cells. I was successful in terms of collecting the plasmid DNA during mini prep, except that the DNA didn’t turn out to be the ones I was looking for. Tomorrow I am going to start over on my PCR and work through the whole process again. Even though not getting the mutant on the first trial, I was able to gain a complete understanding of what I am trying to do, which is really worthwhile after all.      

For the past one week I have read numerous papers on motor proteins. My PI, Dr. Endow, spends about 30 minutes daily with me to discuss the material I have read. Here is what I have learned from reading this particular paper by Dr. Endow.

“Kinesin Motors as Molecular Machines”  by Sharyn A. Endow. BioEssays 25L1212-1219, 2003, Wiley Periodicals, Inc.

The mechanism of how motors convert the energy the energy from ATP hydrolysis to work is yet to be discovered. It is known that motors are able to convert chemical energy from hydrolysis directly into work. ATP hydrolysis leads to a conformation change of the motor protein, and it is this conformation change that provides the potential for protein movement. ATP hydrolysis induces change of motor protein conformation that results in strains, and the spring-like and elastic characteristics of the motor protein has the tendency of converting back to the original position to release the strain, which results in the movement of motor protein.The proposed model of motor proteins is believed to work like a machine composed of a lever, amplifying the conformational change, a hinge, driving the lever, and a latch that regulate the binding of nucleotide. The stalk or the neck linker region is thought to manipulate the direction in terms of how motor proteins move.  

Our lab focuses on the study of motor protein, specifically on the kinesin family. The kinesin family is a group of motor proteins that move along the microtubule filament. Conventional kinesin proteins are characterized by their directionality, moving toward the plus end of the microtubule. Ncd motor protein, however, is a special one that moves toward the minus end of the microtubule. Majority of the kinesin proteins are known to have two motor domains, a neck and a coiled-coil stalk region. Binding with microtubule promotes the hydrolysis of ATP on the catalytic site of motor proteins, and it is ATP hydrolysis that provides energy for motor proteins to walk on the microtubule filaments. My project involves creating a mutant Ncd by alternating the amino acid glycine to aspartic acid. I will conduct motility assays to detect the movement of motor proteins due to its altered conformation.