Mark Botirius

DESCRIBE AND EXPLAIN THE STRUCTURES AND FUNCTIONS OF SEVERAL PROTEINS

Because there seems to be an infinite number of different types of proteins, it was a challenge to select a few out of the vast multitude since each protein serves a purpose related to its structure and function. Although I could choose any protein, I wanted to identify those that stood out from the rest for some reason or another. Therefore, I chose three proteins that I felt were especially meaningful. The first, and the most exciting, are the immunoglobulin proteins because of the unique processes that give rise to them. Following the immunoglobulin proteins is Rubisco, because it is by far the most abundant protein on earth that is part of, what could be considered to be the most important biological process on earth, photosynthesis. For my last choice I was going to select hemoglobin due to its role in every human being as the life sustaining carrier of oxygen, but that would have left me with three globulin proteins, and for variety, I wanted at least one protein that was relatively stationary as part of a membrane. For this, I chose a receptor tyrosine kinase.

Immunoglobulin IgG

Figure 1. The picture on the left is from Austin Community College, and the picture on the right is from the National Center for Biotechnology Information (a national database, similar to doing a BLAST search), which is part of the NIH (National Institutes of Health). On the left, the light chains are depicted in light green, and the heavy chains are depicted in pink. The “C” stands for the constant region, and the “V” stands for the variable region. The antibody binds to antigens at the variable regions. On the right is the ribbon model. The light chains are light brown and turquois, and the heavy chains are pink and blue.

Immunoglobulin G is comprised of two pairs of protein chains that have identical primary structuresi. The smaller chains are called light chains, and the larger chains are called heavy chains. Architecturally, they are composed almost entirely of β pleated secondary structures (as can be seen in the ribbon model) consisting of a diverse amino acid content that is nonetheless held together mostly by hydrophobic interactions and a few covalent disulfide bonds from cysteine residues

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Mark Botirius

(shown in the picture on the left) that together forms a stable tertiary structure. In the center of the ribbon structure there is an area that has neither an α helix or β pleated sheet. Rather, it consists of a flexible hinge, which gives the molecule elasticity that serves to augment its ability to bind to an antigen. Notice that the β pleated sheets of both the heavy and light chains in the ribbon model are connected by single stranded loops. The loops located at the top right and top left of the molecule comprise the areas in the variable regions where the antibody binds to an antigen. When we consider the various structural aspects of this molecule, it is clear that this molecule is structurally adapted to function as a binding molecule. The β pleated sheets give it the necessary strength and stability, and yet the looping single strands give it the needed flexibility to bind strongly to an antigen. Flexibility is also found at the center of the molecule at the hinge.

What is so amazing about this antibody molecule (and also about all antibody molecules in general) is that if you picture the molecule without its variable light and heavy regions on the ends, it is clear that it is an excellent platform for a molecule whose function is to bind a variety of other molecules. As I have just described, it is both structurally stable and yet flexible. It is similar to how a multi-tip screwdriver handle is an excellent platform to turn various screw types. If you need to turn a standard screw, you pop on a standard tip. If you need to turn a Phillip’s screw, you pop on a Phillip’s head. Incredibly, this is sort of what happens in our immune systems. The difference is that our immune systems aren’t making tips to fit a particular screw, they make as many different kinds of tips as possible, in the event they run into a “pathogen screw” that fits their particular tip. The genes that encode the variable regions at the ends of the “Y” on the antibody molecule are composed of many segments. These segments are different for light and heavy chains; however, the principle is the same. For example, the light variable chain segments are comprised of two categories: V and J. There are several different “V” segments, and several different “J” segments. Only one of each is brought together in a developing B cell to create (literally) the gene for the variable region. In other words, the gene that codes for the variable region is created in the B cell through genetic recombination. It turns out, nature has been in the genetic engineering business long before mankind was even a twinkle in the evolutionary eye! In this way, the immune system can theoretically create an antibody within a given population for almost any pathogen in existence, by creating different “tips” (the variable regions on the ends) and attaching them to a standard “platform” (the constant regions).

V1 V2 V3 J1 J2 J3 J4 C

Variable Region Constant Region

Figure 2. The top row represents the region of DNA that contains the various V, J, and C segments. Through DNA recombination, one of each of the V, J, and C segments is brought together to form a contiguous gene represented by the bottom row. Because each gene is different, they each code for a different variable region, which can bind to a different antigen.

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V3 J2 C

Mark Botirius

Rubisco

Because RuBisCo (ribulose 1, 5 bisphosphate carboxylase / oxygenase) is the only known protein that can fix CO2 in higher plants, it is arguably the most abundant protein on earth. Despite its important role in the biochemistry of life, it is a rather slow and inefficient enzyme. On average, RubisCo can only catalyze 3 to ten molecules each second (Cambridge CAPP, 2016). One reason is because RuBisCo, as its unabbreviated name indicates, is both a carboxylase and an oxygenase. Although it will preferentially bind CO2, it does so only slightly, and so in the presence of oxygen it not only attaches carbon to RuBP, it will attach oxygen as well, which is a

costly move for the plant biochemically. To process the oxidized RuBP molecule (called 2-phosphoglycolate) the plant ends up losing CO2.

The primary structure of this protein is also highly varied, and the secondary structure, as depicted in figure 3, consists of both α helices and β pleated sheets in addition to loops. An analysis of the secondary structure reveals that the α helices and β pleated sheets located in the center are hydrophobic, while the loops located along the periphery and in the center hole are mostly hydrophilic. This allows RuBP access to the active sites which are located on the large subunits of

the molecule towards the center. These active sites are constructed of β barrels surrounded by α helices. The β barrels are the catalytic centers of the active site that hold a Mg2+ ion in the catalytic pocket by interacting with the polar charged amino acids histidine and lysine. This is a good example of the relationship between structure and function. The creation of a six carbon intermediate sugar from RuBP is accomplished via a charged metal ion. This is the reason that we find charged polar amino acids in the catalytic site. When CO2 enters the active site, it attaches to the lysine residue, which destabilizes the metal ion that results in a conformational change that brings the RuBP molecule and CO2 molecule together, catalyzing the reaction that

forms a six carbon sugar. (PNAS, 2017) (Goodsell, 2017)

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Figure 3. This is a ribbon model for the RuBisCo protein found in a spinach plant. It is composed of 8 large subunits and 8 small subunits.

Figure 4. Ribulose 1,5 bisphosphate carboxylase/oxygenase active site

Mark Botirius

Tyrosine Kinase Receptor

Lastly, I chose a receptor protein because it demonstrates nicely the connection between structure and function; since there is a distinct difference between those structures, for example, that are inside the membrane of a cell in contrast to those that are sticking out from the ends. Tyrosine kinase receptors are so named because they are a signaling molecule that, when bound to their particular ligand, phosphorylate (enzymes that phosphorylate other molecules are called kinases) their tyrosine residues. RTK’s are amphipathic integral membrane proteins that consist of three domains: an outer domain that protrudes outside of the cell and serves to bind ligands, a middle domain that is situated inside the cell membrane, and an

inner domain that protrudes into the inside of the cell that serves to deliver the signal received from the ligand. This is an excellent example of the connection between structure and function. The outer ends of the molecule are in contact with the aqueous environments that exist both within and without the cell. The middle section, has a primary structure made up entirely of non-polar amino acids, that have adopted an α helical secondary structure. This nonpolar middle section is in direct contact with the non-polar phospholipid tails that make up the intermembrane space.

This, along with an α helical secondary structure, secures the protein in the membrane.

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Figure 5. An NCBI rendered ribbon model of a tyrosine kinase receptor

Figure 6 This is a tubular model of the protein depicted in figure 5. The canonical sequence shown is the actual sequence of the protein, and the highlighted areas depict the hydrophobic region of the molecule to clearly show the connection between the structure and function of the amino acids.

Mark Botirius

If you look carefully at the actual canonical sequence, you will notice that the tyrosine residues are missing. That is because this is the actual sequence from the paper shown in the NCBI picture in figure five, which focused specifically on the transmembrane region. The shortened sequence also made it easier for me to put the amino acid sequence in the text, so that I could more clearly demonstrate the hydrophobic structure – function connection. The overall receptor function of the molecule is more clearly shown in the following figure.

As can be seen in the figure above, when a ligand binds to receptor domains of the proteins, a conformational change brings the to two dimers together. As a result, the two dimers actually phosphorylate each other, activating them. The activated kinases then phosphorylate another molecule, which, when activated phosphorylates yet another molecule down the cascade. One possible result of the conclusion of the cascade is the activation of some transcription factor that influences genetic expression. It is no accident that the amino acid tyrosine is found inside the cell. Tyrosine, serine, and threonine are almost exclusively the active residues in the phosphorylation activities of proteins. Why? Because these are the only amino acids with an OH group as part of their side chains, and the hydroxyl group is where the phosphates are added (or removed). Likewise, many of the extracellular domains of kinases have polar and/or charged residues. The reason is because they often need to be able to bind with ligand molecules with an affinity that is greater than that of uncharged or nonpolar molecules.

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Figure 7 The two blue structures are the two RTK domains pictured as pink and blue in figure 5. This picture is from “Membranereceptors.com

Mark Botirius

EXPLAIN AND SHOW HOW THE REACTIONS OF THE CENTRAL METABOLISM (GLYCOLYSIS, TCA CYCLE, OXIDATIVE PHOSPHORYLATION) INTERACT WITH ONE ANOTHER

After careful consideration, I have concluded that glycolysis is the most likely place to begin, because its carbon starting material (glucose) is not provided by the other two pathways, and of the three, it appears to have the fewest molecules fed into it from the others. My biggest concern, is that my answer is cohesive, organized, and clear. To ensure this, I have decided that the best way is to address each reaction separately, step by step, and ask two questions. 1. What, if anything, did the other two reactions contribute to this step? 2. What, if anything, does this step contribute to the other two?

Glycolysis

1. Glycolysis begins with glucose as its starting material. Glucose is phosphorylated by the enzyme hexokinase, using a molecule of ATP in the process.

a. What, if anything, did the other two reactions contribute to this step?

The glucose came from sources outside the three reactions under consideration, so they did not contribute the glucose. The ATP, theoretically, could have come from any of the three, since all three produce ATP. Since glycolysis also produces ATP, I would say that most likely, the ATP came from glycolysis itself.

b. What, if anything, does this step contribute to the other two?

The product from this reaction (Glucose 6 phosphate) is not used in either the TCA or ETC reactions. So nothing is passed on to the other two. Since the ETC needs ADP to make ATP, it is possible that the ADP is contributed to oxidative phosphorylation.

2. In the next step in glycolysis, fructose 6 phosphate is produced from glucose 6 phosphate via the enzyme phosphoglucose isomerase. The starting material (glucose 6 phosphate) came from the previous step, and the product is not used in either the TCA or ETC reactions. Therefore, the answer to both questions is none.

3. Next, fructose 6 phosphate is phosphorylated by the enzyme phosphofructokinase to produce fructose 1,6 bisphosphate. The answers to both questions are the same as in step one, that is, none.

4&5. Next, fructose 1,6 bisphosphate is split into two three carbon molecules. One is glyceraldehyde 3-phospate, and the other is an intermediate metabolite, dihydroxyacetone phosphate. Dihydroxyacetone phosphate is then isomerized into glyceraldehyde 3-phosphate. So overall, this reaction produces 2 molecules of G3P. It should be noted, that the products of this reaction (G3P) are intermediates in other reactions not under consideration here, such as the Calvin cycle. However, the answers to both questions at this point is still none.

6. In this step, both molecules of G3P are oxidized by NAD+ reducing two molecules of NAD+ to NADH + H+. The product is 1,3 bisphosphoglycerate.

a. What, if anything, did the other two reactions contribute to this step? P a g e 6 | 10

Mark Botirius

In the presence of oxygen, only the ETC produces NAD+. The other two reactions, glycolysis and TCA, consume it. In the absence of oxygen, however, the end product of glycolysis, pyruvate, can be reduced to produce NAD+. Therefore, the NAD+ could have come from the ETC or fermentation.

b. What, if anything, does this step contribute to the other two?

The NADH molecules carry electrons that are used in the ETC reactions to drive protons into the inter membrane space of the mitochondrion to produce a proton gradient. It is possible, the H+ molecules also help contribute to the gradient.

7. In this step 3-phosphoglycerate is produced from 1,3-bisphosphoglycerate producing one two molecules of ATP from ADP (remember, everything from steps 4 & 5 involve two molecules). I consider these molecules of ATP to be what was needed to bring the glycolysis balance to 0. Therefore, the answer to questions a and b are none.

8, 9, & 10. None of the intermediate products in these steps are used in the other two reactions. The only thing worth noting is that two more molecules of ATP are produced from ADP. Since ADP results from the utilization of energy, it is pretty much ubiquitous, and therefore there is no real need to connect it to any particular reaction. Lastly, the end product of glycolysis, pyruvate, is used in the TCA reaction.

The TCA Cycle

A careful look at the TCA cycle reveals that it is essentially little more than an electron carrier producing reaction. The only exception is that it uses one GDP molecule to produce GTP. It neither produces or uses either ATP or ADP. Furthermore, it’s intermediate metabolites are not used by glycolysis or the ETC. Therefore, we need to only summarize its uses of NAD+, FAD and GDP to account for its relationship to glycolysis and the ETC.

The TCA produces NADH from NAD+ in the following reactions:

1. Oxidizing pyruvate to produce Acetyl CoA

2. Oxidizing isocitrate to produce α ketoglutarate

3. Oxidizing α ketoglutarate to produce succinyl CoA

4. Oxidizing malate to produce oxaloacetate

All of the NAD+ for these reactions was supplied by the ETC. Glycolysis (fermentation notwithstanding) and the TCA reactions do not produce NAD+. The NADH produced is used by the ETC.

The TCA cycle also produces the electron carrier FADH2 from FAD when it oxidizes succinate to produce fumarate. Again, the FAD is supplied by the ETC, and the FADH2 is used by the ETC.

Lastly, a molecule of GDP is phosphorylated when a molecule of succinate is produced from succynil CoA. This is the only place that we find GDP, and it is used extensively throughout the cell, not just in this reaction.

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Mark Botirius

Oxidative Phosphorylation

The oxidative phosphorylation cycle consists of a series of membrane bound electron carriers that are arranged in order from the one with the greatest redox potential to the one with the least. NADH and FADH2 give their electrons to the carriers with the greatest potential that can accept their electrons. These carriers then use the energy to shuttle protons across the membrane and then pass the electrons to the next carrier, which does the same. Finally, at the end of the chain, the electrons are passed to an oxygen. Therefore, the ETC uses the NADH and FADH2 produced by glycolysis and the TCA reactions to make NAD+ and FAD that is used by glycolysis and the TCA. The protons are used by ATP synthase to make ATP from ADP. ADP is produced throughout the cell. The following figure summarizes these interactions.

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Glycolysis

The Citrus Acid Cycle Oxidative Phosphorylation

NAD+ FAD

NAD+

NADH ADPPyruvate

ATP

NADH FADH2

ATPGDP GTP

NAD+ from fermentation

Mark Botirius

References(2016, September 25). Retrieved from Cambridge CAPP:

https://cambridgecapp.wordpress.com/improving-photosynthesis/rubisco/

(2017, September 25). Retrieved from PNAS: http://www.pnas.org/content/109/46/18785.full#F4

Goodsell, D. (2017, September 25). Retrieved from PDB101: http://pdb101.rcsb.org/motm/11

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i For example, the canonical sequence for the heavy chain pictured in the ribbon model is: 1 qvqlvqsgae vkkpgasvkv scqasgyrfs nfvihwvrqa pgqrfewmgw inpyngnkef 61 sakfqdrvtf tadtsantay melrslrsad tavyycarvg pyswddspqd nyymdvwgkg 121 ttvivssast kgpsvfplap sskstsggta algclvkdyf pepvtvswns galtsgvhtf 181 pavlqssgly slssvvtvps sslgtqtyic nvnhkpsntk vdkkaepksc dkthtcppcp 241 apellggpsv flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk 301 preeqynsty rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt 361 lppsrdeltk nqvsltclvk gfypsdiave wesngqpenn ykttppvlds dgsfflyskl 421 tvdksrwqqg nvfscsvmhe alhnhytqks lslspgk