Episode 7: Snapback to Reality

This week a Japanese music video has taken flight. PPAP has taken the internet by storm. Pen Pineapple Apple Pen is simply the best. Memetastic. This old man is balling out of control.

Hours after axing like 12 sources and literally gathering another 14 from scratch over the weekend (exaggerating). My paper looks better.

My research question is 

Do certain changes at the conserved positions of LAC1 significantly change the function and dynamics of the protein?

1. Scope: My scope is narrowed to LAC1 and the position I will mutate is at 52 on the LAC1 chain. I chose 52 because it is the most conserved position on the protein according to one of my studies. I will consult with my professor on the position number. My scope is manageable, I can even look at more positions in different levels of conservation.

2. Definitions: I need to define, LAC1, conserved, dynamics. I do define them in my literature review.

Certain proteins block the DNA transcription. These molecules are called repressors. The function of the genes that lac suppresses is to code for proteins that break lactose into glucose, galactose, and other sugars. Lac 1 is a repressor. Lac repressor prevents the creation of Beta-galactosidase, which is the enzyme that performs the first step in lactose metabolism, making glucose and galactose. Conserved positions are segments of a protein that are essential to the structure of a protein. Dynamics are the movements of the protein. An assumption may be that conserved positions are important to look at, but its justified in the definition.

3. Variables: The function and dynamics are variables that I will analyze

4. Researchability: 100%

I am going to work with the people who created DFI, and I will use DFI to analyze the proteins. DFI will show the changes in the dynamics of LAC1

5. Gap: LAC1 has not been computationally analyzed with DFI. And understanding the dynamics of LAC1 is significant because of its function of breaking down lactose. Also DFI has not been used that often, and using it again to possibly justify or corroborate with other methods can help support the method.

6. Significance: The larger real world implications include solving protein folding diseases, and understanding how changing protein code leads to human diseases.

And with that I can finally sleespflpcm,va.gagaregf.....

12 hours of sleeping later, I am posting this blog.

Episode 6: Into the Darkness

The meme of the week is definitely gonna be grinds my gears. A classic from family guy where people express their discontent.

Subtopic 1: Protein engineering and folding history.

Start with this because it sets up the background and creates the significance for my research. This topic will explain the goals of protein folding and how it has progressed through the years.

1. Protein engineering’s end goal is to make new proteins or new functions out of existing proteins or from scratch
1. Directed Evolution is a way to achieve that goal
1. Lutz discusses the process of directed evolution and the history of directed protein evolution. They talk about the computational tools for the evaluation of protein data sets. The methodological advances in semi-rational enzyme engineering and de novo enzyme design in recent years provide researchers with powerful and effective new strategies to manipulate biocatalysts. Directed evolution represents an obvious strategy to improve existing designer enzymes and to potentially “break” the performance cap.
2. Packer talks about pioneering studies in the field of directed evolution sought to improve the wild-type activity of enzymes through the enhancement of solubility, thermostability, affinity for substrate or catalytic turnover in simulations. The future of new directed evolution studies is bright.
2. In order to create new proteins, we must correctly predict the structure of the protein, which is no easy task.
2. 60 years ago, there were no discernable methods to figure out the specifics of protein folding, through Moore’s Law the methods will be more advanced in the future
1. Dill talks about Moore’s Law and the benefits of folding in the future, including the possibility of solving folding failures like Parkinsons and Alzheimers
3. Currently, there are many methods but experimentation and computer modeling are leading the way.

Subtopic 2: Different prediction methods Computer Simulation vs Experimentation.

1. Experimentation in labs is more Accurate
1. This only results in a small amount of structures because it is time consuming (2500)
1. Kumar
2. Computer analysis methods are less accurate now, but have the potential to create much more structures in a much faster manner in the future
1. They are also currently the most accessible form of analysis
1. Need to back this up
3. Thus computer models are the future of this field
1. CASP competition
1. Recently the computer based prediction models are more accurate than other methods
1. Kumar and CASP
2. Because computer based models are the more effective long term solution, I will be using them
2. REMD, SIFT, FIRST
1. I need to look into this some more and compare these methods, I just found some articles for this
2. FIRST, and REMD
1. De Graff
1. Crack Propagation
2. Thorpe
1. New methods for protein folding by using short-range forces in proteins modeled by constraints. Forces included in the analysis are the covalent bond-stretching and bond-bending forces, salt bridges, and hydrogen bonds. They used colored mapping to provide a visualization of the motion of the protein.
2. FIRST stands for Floppy Inclusion and Rigid Substructure Topography.

Subtopic 3: LAC1 Repressor, why look at it?

1. Definition of LAC1 Repressor
1. The crystal structure of the lac repressor is a bent structure with all 4 of the DNA binding portions pointing in one direction.
2. The DNA sequence has 3 lac repressor recognition sites within 500 base pairs. In the quaternary formation of the repression loops, the repressor interacts simultaneously with 2 genomic DNA sites.
3. Lewis
2. The repressor is important because it suppresses the coding for proteins that break down glucose, galactose, and other sugars.
1. Prevents the creation of Beta-galactosidase, which is the enzyme that performs the first step in lactose metabolism, making glucose and galactose. Galactoside acetyltransferase, which actually doesn't really do much in lactose metabolism (its unclear). And lactose permease transports lactose through membranes.
2. LAC1 plays a key role in lactose breakdown, understanding exactly why the protein functions the way it does will provide insight into diseases such as Lactose intolerance.
3. Goodsell from the Protein Database
4. Possibly engineer the LAC1 into not repressing those enzymes

Subtopic 4: Conserved vs Nonconserved positions

1. Certain parts of protein are essential to function, others are not
1. There is a dispute on whether or not all segments of the protein are essential to function
2. Conserved positions control major folding points that change the entire structure. Nonconserved positions control less important points in the protein that do not contribute to major structural problems.
1. However the function may be affected by Nonconserved position
3. Over many years scientists have attempted to change nonconserved positions to fine tune function.

Subtopic 5: The significance of Structure for Function

1. The structure of the protein is essential for understanding the biological role of the protein. Structural genomics aims to structurally characterize most protein sequences by a combination of experimentation and prediction
1. Baker
2. Could be placed after Subtopic 1

Combining both the significance of protein folding and prediction with the protein of LAC1 leads us to investigate the importance of Conserved vs nonconserved positions. Thus the question: Do nonconserved positions, when mutated, affect the function of the protein more significantly than conserved positions in LAC1.

Purpose: Understand definitions and the methods for testing LAC1. And understanding the future implications of the results whether it corroborates common evidence or not.

So I got a lot of subtopics, but I feel like the order could be moved around, if you could help me out that would be great. Help me out friends.

Episode 5: RIP Keenan Allen

Fantasy football just started, and my best wideout Keenan Allen just tore his ACL. I actually can't stand this, also I hate the snapper for the Cardinals right now. The meme for this week is Squidward Dab. This a classic where a dude in a squidward costume dabs in the middle of a children's parade. I actually can't stop laughing every time he hits the dab. It's lit. Anyway for this task its the Academic Conversation.

This week on Academic Conversation Death match, in corner A we have Swint-Kruse, L. with his work Using Evolution to Guide Protein Engineering: The Devil IS in the Details. In corner B we have Zahn, H. with his work, Subdividing Repressor Function: DNA Binding Affinity, Selectivity, and Allostery Can Be Altered by Amino Acid Substitution of Nonconserved Residues in a LacI/GalR Homologue. The topic of today's death match is whether or not mutations at non-conserved residues significantly impact protein function.

SK: Protein Engineering has always attempted to edit nonconserved residues and lead us towards new proteins with new functions. However, the scientific community has been blinded with this primitive assumption. I have found in my study that mutations at nonconserved positions could completely impede the function of proteins, just like mutations at conserved positions completely alter the folding of the protein.

Z takes a blow to the right side of his head.

Z: While the community has been dead set on nonconserved positions having a minor impact on protein folding, there is a reason that assumption is true. It would only make sense that insignificant coding regions in DNA won't change the overall structure of protein created. In my study, we tested several different residue sites, both conserved and nonconserved. We found a varied range effects on the function of the protein. Surprise? I think not. There is always a varied scale.

SK kneels to the floor and gets kicked to the floor.

SK: You must at least consider my studies, because they show a flaw with in silico experimentation. There are limitations associated with the prediction models and mutations at all locations may not even be altering the structure. But rather the folding itself may be inaccurate.

Z: That is a point we can agree on. We must take into account the limitations of our style of experimentation and work to increase the accuracy of these programs.

SK and Z shake hands, showing the true power of academic conversation.

But Z backstabs SK and the side I supported prevails.

(I wrote most of this last night)

Episode 4: I'm Ashwath, and this is my research blog

The meme this week is pretty fresh, only 20 days old. It's the I'm Rick Harrison and this is my pawn shop. I work here with my old man and my son, Big Hoss. Everything in here has a story and a price. One thing I’ve learned after 21 years – you never know WHAT is gonna come through that door. I don't understand myself why this is so funny, but the internet is going crazy replacing this classic intro with other people. I’m Harambe, and this is my zoo enclosure. I work here with my zoo keeper and my friend, Cecil the lion. Everything in here has a story and a price. One thing I’ve learned after 21 years – you never know WHO is gonna come over that fence. This cracks me up every time, but anyways back to AP Research. I've read more sources, so that's cool.

First, I'm Ashwath, and this is my research blog. I type here with my dax and my teacher, Mrs. Haag. Everything in here has a story and a meme. One thing I've learned after 4 weeks - you never know WHAT is gonna come through the works cited section. OK I'm done. But Literature reviews am I right? For my literature review, I need to set up the significance, any relevant information, and my gap in the research. So to build the significance of my protein research, I need to set up why finding a difference in conserved mutation function and non conserved mutation function matters. To show this I plan to talk about the history behind protein folding, and why it has always been a struggle to properly predict the folds a protein makes. Whether these mutations change the function (folds) can lead to further research techniques for large proteins like LAC1 in the future. There will probably have to be a large amount of defining or watering down in the literature review because a science background is practically required to decipher the research papers. But hey if I am able to understand it (which I am), I can translate into more layman terms. But as of now I don't know too many of the terms that need to be defined. Finding a gap in my research is relevant for A) moving towards a more efficient computer based prediction model over lab based procedure that are long and expensive and B) LAC1 which could help guide research in understanding the ways to fix the folding problems associated with its diseases. BUT the number 1 goal of my lit review is to have the reader understand what I am writing.

Second, I'm Ashwath, and this is ... OK I'm actually done. But an important source is probably the one from the Protein Data Base Databank describing the LAC1 protein function. This is probably the most important gap I have, which is the uniqueness of the LAC1 repressor. It is not only linked to specific diseases such and Lactose intolerance, it is also a large protein sequence, which computer simulations have struggle with. The source described the function of the genes that LAC1 protein represses. The genes code for proteins that break lactose into glucose, galactose, and other sugars. The LAC1 repressor prevents the creation of Beta-galactosidase, which is the enzyme that performs the first step in lactose metabolism, making glucose and galactose. It also doesn't produce Lactose permease transports lactose through membranes. These details describe its function. The crystal structure of the lac repressor is a bent structure with all 4 of the DNA binding portions pointing in one direction, showing the largeness of the structure, 333 amino acids long. So yeah this source is pretty useful. This source doesn't have a real academic conversation surrounding it, but there is more conversation with computer simulations vs. lab results, and conserved/non-conserved positions existing or not. That is where the other gaps will come, however I think this is the strongest gap I have and thus I talked about it. I have the sources describing my methods and the computer simulations on the way, so I'll get some more controversial foundational sources soon.

Third, I'm John Oliver and this is my comedy show. I work here with my writers and my inspiration, Harambe. Everything in here has a story and a price. One thing I’ve learned after 2 years – you never know WHAT is gonna be corrupted. Ok actually I'm done. I love John Oliver and I watch him every Monday morning, even though I don't agree with him every time. Discussing this baller in class is chill with me, although I hope we don't get too hung up on him to keep paper on time. I watched the charter school segment and I agree with Oliver on most points, but I am happy to argue from a BASIS point of view.

Fourth, IM BILLY MAYES AND THIS IS MY OXI-CLEAN STAND. I WORK HERE WITH MY OLD MAN, MR. CLEAN, AND MY SON, BARRY B. BENSON. EVERYTHING HERE HAS STAIN REMOVING POWERS.. AND A LARGE PRICE. ONE THING IVE LEARNED AFTER 21 YEARS- YOU NEVER KNOW WHAT LIQUID WILL RUIN YOUR CARPET/CLOTHES/ETC NEXT. Actually I ran this meme into the ground at this point. (871(theres defn 400 relevant words in here i promise))