BIOL368/F14:Isabel Gonzaga Week 6

From OpenWetWare

Jump to: navigation, search

Contents

STAR Biochem DNA Glycosylase

Methods and Results

Method taken from DNA Glycosylase Exercise

StarBiochem was downloaded and ran using a Java applet. I hit start, then selected: Samples > Select from Samples. Within the Amino Acid/Proteins > Protein tab, select “DNA glycosylase hOGG1 w/ DNA – H. sapiens (1EBM)”

  1. The hOGG1 structure contains both DNA and protein. Can you differentiate between the DNA and protein components? How did you distinguish the DNA from the protein?
    • The DNA Sequence forms an alpha helix on the side of the page. This structure is very easily recognizable. because of the planar base stacking. the protein complex contains many side chains and has a repetitive pattern of peptide bonds (featuring nitrogen and carbon atoms)
      Figure 1.Image of isolated amino acid, Cysteine, as part of the hOGG1 protein. The sulfur atom is pictured in yellow.
      Figure 1.Image of isolated amino acid, Cysteine, as part of the hOGG1 protein. The sulfur atom is pictured in yellow.
  2. hOGG1 contains multiple sulfur atoms.Identify the name and sequence number of one of the amino acids in the structure that contains a sulfur atom.
      • Answer: Cysteine, position 146. This is located in the side chain of the amino acid.
      • Procedure:
        • I pointed to a yellow (sulfur) Atom in structure. A small box appeared on top of the mouse cursor indicating the name of the amino acid and its position in the amino acid sequence
        • From the top menu, I selected: View> View Specific Regions / Set Center of Rotation. I selected the CYS at position 146. I moved the VDW Radius slider all the way to the left (1 Van der Waals radii) and zoomed in. Unchecking the back bone box (leaving side chain), I saw that it must be a part of the side chain.
  3. Next, I explored the primary structure of the hOGG1 protein. The hOGG1 protein consists of 325 amino acids. List the 13 amino acids numbered 105 through 117 in order.
    • Answer: Threonine, Leucine, Alanine, Glutamine, Leucine, Tyrosine, Histidine, Histidine, Tryptophan, Glycine, Serine, Valine, Aspartate
    • Procedure: I reset the structure. Hit Protein -> Primary tab and found the 3 letter codes for amino acids 105-117.
  4. Within a protein chain, amino acids form local structures called secondary structures (Reference page).
    • a) Explore the secondary structures found in hOGG1. Are helices, sheets or coils present in hOGG1? Describe the color that represents the secondary structures you observe.
      • Answer:
        • Alpha Helices: Magenta
        • Beta Sheets: Yellow
        • Random Coil: Blue
      • Procedure: I clicked on the secondary tab and checked the box beside the desired structure (e.g. helices, sheets and coils) and moved the structure size slider to the right to increase the size, one by one.
    • b) Amino acids 105 through 117 fold into one of the secondary structures. Which secondary structure do they fold into?
      Figure 2.Alpha helix secondary structure formed by amino acids in positions 105-117 in hOGG1 protein
      Figure 2.Alpha helix secondary structure formed by amino acids in positions 105-117 in hOGG1 protein
      • Answer: Alpha Helix
      • Procedure: In the View Specific Regions window, I went to the Protein - Secondary tab and clicked the All button. In the amino acid sequence window, I selected 105-117 and moved the VDQ radius slider to the left to show only the selected amino acids in the viewer.
  5. Then I explored the relationship between DNA glycosylase’s structure and one of the several types of amino acids that contribute to hOGG1’s overall shape, its tertiary structure (Reference page).
    • a) Negatively charged amino acids are hydrophilic (Reference page). Are the negatively charged amino acids located on the inside (buried) or outside (exposed) of this protein? What does that suggest about the cellular environment surrounding this protein, is it hydrophobic or hydrophilic? Explain your answer.
      Figure 3.Negatively charged amino acids stick to the outside of the folded hOGG1 protein, allowing the exterior to be more hydrophilic and interior to be hydrophobic.
      Figure 3.Negatively charged amino acids stick to the outside of the folded hOGG1 protein, allowing the exterior to be more hydrophilic and interior to be hydrophobic.
      • Answer: Negatively charged atoms are on the outside of the protein. This means that the cellular environment must be hydrophilic, because it interacts with the polar, charged atoms.
      • Procedure: I reset the structure. Then I clicked on the tertiary tab, and moved the atoms size slider to the left to decrease size for all atoms in the protein. I clicked on the negatively charged/acidic button and moved the atom size slider to the right to increase the atom size for the negatively charged/acidic amino acids.
  6. Then I explored how DNA glycosylase interacts with DNA to recognize the damaged DNA base within its sequence. DNA is composed of four bases: Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). In this particular structure, the hOGG1 protein is bound to a segment of DNA that contains an oxidized guanine.
    Figure 4.DNA double helix structure within the hOGG1 protein
    Figure 4.DNA double helix structure within the hOGG1 protein
    • FIrst, I made the DNA more visible
      • I reset structure, then clicked on the Nucleic Acids tab to move the Atoms Size slider to the right (35%), increasing the size of the DNA segment.
      • In the Protein - Primary tab, I moved the Atoms Size slider completely to the left and the Bonds Translucency slider to the right (95%) to minimize the appearance of all the amino acids in the hOGG1 protein.
    • Second, I looked at how DNA bases are oriented within the double helix. Each base is attached to a sugar and phosphate backbone to form a complete nucleotide. Within the double helix, a base within one strand pairs with another base from the opposite strand by hydrogen bonding forming a base pair: Adenine (A) base pairs with Thymine (T) and Cytosine (C) base pairs with Guanine (G).
      • Nucleic Acids tab: unchecked phosphates and sugars. I moved the Atoms Size slider to the right (70%) to increase the size of the bases while leaving the size of the phosphates and sugar nucleotide components intact. This allowed me to count the bases more easily. Then, I clicked on the Non-Peptide tab. I moved the Atoms Size slider completely to the right to increase the size of the atoms in the oxidized guanine ([8OG]25).
    • Questions
      • a) How many DNA base pairs can you count within this double helix? How many bases are unpaired?
        • Answer: 14
      • b) How many DNA bases are unpaired (not paired to its partner on the other strand)?
        • 2 unpaired bases
      • c) Is the oxidized guanine base paired or unpaired? Describe the position of the oxidized guanine with respect to the hOGG1 protein and the double helix. What does this suggest about the mechanism that hOGG1 uses to identify damaged DNA bases?
        • The oxidized guanine base is unpaired. the base is positioned away from the double stranded DNA helix. This suggests that the hOGG1 identifies damaged bases based on their orientation relative to the rest of the molecule. If it doesn't fit exactly or sticks out or is oriented away, then the protein will come and repair the damaged site.
  7. Certain amino acids within hOGG1 form contacts with the DNA and are able to recognize if a guanine base has been damaged by oxidation. Where are you more likely to find the amino acids that recognize damaged guanine bases within hOGG1, in Helix 1 or Helix 16? Explain why.
    Figure 5. Alpha helix 1 (designated by arrow) in relation to the location of the double stranded DNA
    Figure 5. Alpha helix 1 (designated by arrow) in relation to the location of the double stranded DNA
    Figure 6. Alpha helix 16 in relation to the location of the double stranded DNA
    Figure 6. Alpha helix 16 in relation to the location of the double stranded DNA
    Figure 7. Alpha helix 16 with side groups in relation to the location of the double stranded DNA. The amino acid residues directly interact with the unpaired Glutamine, showing it's ability to recognize the damaged site.
    Figure 7. Alpha helix 16 with side groups in relation to the location of the double stranded DNA. The amino acid residues directly interact with the unpaired Glutamine, showing it's ability to recognize the damaged site.
    • Answers:
      • Helix 16 recognized more damaged guanine bases. This is 310-324. The sequences are as follows:
        • Helix 16: Tyrosine, Alanine, Glycine, Tryptophan, Alanine, Glutamine, Alanine, Valine, Leucine, Phenylalanine, Serine, Alanine, Aspartate, Leucine, Arginine
        • Helix 1: Threonine, Proline, Alanine, Leucine, Tryptophan
      • The damaged guanine base is oxidized, and now has a carbonyl on the nitrogenous ring. Helix 16 is better able to recognize this, because in protein folding, it is located closer. Additionally, helix 16 has more polar side chains that directly interact with the oxidize guanine. This includes Glutamine. The phenylalanine in the side chain is aromatic and is attracted to the aromatic nitrogenous rings in the guanine base, as it it 'stacks' on top of it due to the overlapping pi bonds. The 15 amino acids composes a larger, bulkier chain. This is thus effective in recognizing damaged DNA, particularly if the nucleotide base is sticking outwards on the side of the helices. Overall, the structure and amino acid side chain activity allows Helix 16 to be more efficient in determining bases damaged by oxidation.
    • Procedure:
      • In the Protein - Secondary tab, I clicked the All button. In the Sequence Window, I selected the amino acids within Helix 1 and increased the size of Helix 1 by moving the Secondary Structures size slider to the right. I repeated this for Helix 16.
      • I then examined if the side chains in Helix 1 or Helix 16 contacted the oxidized guanine ([8OG]25). I clicked on the Protein - Primary tab, Selected the amino acids that make up Helix 1 in the Sequence Window, Unchecked backbone, and moved the Atoms Size slider to the right to visualize the side chains of the amino acids Helix 1. Then I repeated this for Helix 16
  8. hOGG1 is able to distinguish very efficiently between guanine and its oxidized counterpart, 8-oxoguanine. This represents a formidable task given that the oxidized nucleobase, 8-oxoguanine, differs by only two atoms from its normal counterpart, guanine (positions 7 and 8). The following structures illustrate hOGG1 bound to either an 8-oxoguanine or a guanine nucleobase: “1YQR” (8-oxoguanine) and “1YQK” (guanine). I compared these two structures to understand how hOGG1 can effectively distinguish between 8-oxoguanine and guanine, using the diagram under the [| Reference section] for details.
    Figure 8. Interaction between guanine and 1YQK in 1YQK structure of the hOGG1 protein
    Figure 8. Interaction between guanine and 1YQK in 1YQK structure of the hOGG1 protein
    Figure 9. Interaction between 8-oxoguanine and glycine 42 in 1YQR structure of the hOGG1 protein
    Figure 9. Interaction between 8-oxoguanine and glycine 42 in 1YQR structure of the hOGG1 protein
    • Procedure
      • In the top menu, I selected 'Samples' > 'Select from Samples'. Within the Amino Acid/Proteins - Protein tab, I selected “DNA glycosylase hOGG1 w/ DNA containing oxoG - H. sapiens (1YQR)”. “1YQR” is the four character ID for this particular structure.
        • I repeated these steps to open the “DNA glycosylase hOGG1 w/ DNA containing G - H. sapiens (1YQK)” structure. Glycine 42 in hOGG1 directly interacts with the base portion of 8-oxoguanine. This interaction is crucial for detection of oxidative damage of guanine bases.
        • In the Protein - Primary tab move the Atoms Size slider completely to the left and the Bonds Translucency slider to completely to the right to minimize the appearance of all the amino acids in the hOGG1 protein.Click on the Nucleic Acids tab. Click the oxidized guanine ([8OG]23:C) and increase its size by moving the Atoms Size slider completely to the right. In the Protein  Primary tab, click glycine 42 and increase its size by moving the Atoms Size slider completely to the right
    • Questions
      • a) Which atom in the base portion of 8-oxoguanine is directly contacting glycine 42? Draw this interaction (use the chemical structures within this question and the amino acids structures found in the Reference page).
        • The hydrogen from 8-oxoguanine
      • b) What does the interaction between 8-oxoguanine and glycine 42 indicate about how hOGG1 differentiates between 8-oxoguanine and guanine?
        • The carboxyl terminal from glycine hydrogen bonds with the a hydrogen on the nitrogenous base of the 8-oxoguanine. In guanine, there is a lone pair on the nitrogen, so it is unable to react I'm this way. Glycine is likely relevant here because it is the smallest structure, and thus is able to interact at this level with little hinderance.
      • c) Compare the general location and orientation of guanine and glycine 42 in the “1YQK” structure with that of 8-oxoguanine and glycine 42 in the “1YQR” structure. How do the location and orientation of these two nucleobases and glycine 42 differ in these structures? Explain how this comparison adds to your understanding of hOGG1’s ability to discriminate between 8-oxoguanine and guanine.
        • Glycine in both structures are generally located in the same area. However, glycine in the 1YQK structure is directly hydrogen bonding with the 8-oxoguanine. The carboxyl group on the amino acid bonds to the hydrogen on the nitrogenous ring. The orientation is thus different for the 1YQR structure, as the hydrogen on the glycine is more oriented towards the oxygen on the nitrogenous ring. This demonstrates how hOGG1 is able to shif it's folding in higher levels dependent on the ionizability of mutated DNA molecules. Thus, amino acid structure and characteristics become very important in determining reactivity and recognizing key factors.
    • Procedure
      • First I reset the 1YQR structure.
      • In the Protein - Primary tab, I moved the Atoms Size slider completely to the left and the Bonds Translucency slider to completely to the right (95%) to minimize the appearance of all the amino acids in the hOGG1 protein.
      • I clicked on the Nucleic Acids tab. Then clicked on the oxidized guanine ([8OG]23:C) and increased its size by moving the Atoms Size slider completely to the right.
      • In the Protein - Primary tab, I clicked glycine 42 and increased its size by moving the Atoms Size slider completely to the right.
      • Repeated these steps with the “1YQK” structure, but instead of increasing the size of the oxidized guanine, I increased the size of guanine at position 23.
  9. Propose an experiment to test whether phenylalanine 114 plays a role in the detection and repair of 8-oxoguanine nucleases
    • Answer: To test whether phenylalanine 114 plays a role in detecting and repairing 8-oxoguanine nucleases, an experiment should be conducted to compare the mutation rate of 8-oxoguanine nucleases between two versions of DNA Glycosylase - one containing phenylalanine 114 and the other without. If future generations of the cell using DNA Glycosylase without phenylalanine 114 contain mutations or are inactive, then phenylalanine is necessary for function. Mutations can be detected using Southern blotting techniques.

Cn3D

  1. Cn3D was downloaded from the NCBI website
  2. The 1ebm 3D structure was downloaded by clicking 'view in CD3' and saving the file to the hard drive
  3. Cn3D software was used to render and color the structure. Answer the following:
  4. Questions:
    Figure 10. The three domains of the glycolysase protein as viewed in Cn3D using the coloring shortcuts by domain feature. The domains are pictured in brown, pink and blue.
    Figure 10. The three domains of the glycolysase protein as viewed in Cn3D using the coloring shortcuts by domain feature. The domains are pictured in brown, pink and blue.
    Figure 11. The glcolysase protein as viewed in Cn3D, pictured similarly do Figure 4. This viewing was done by using the ball and stick model and coloring by Molecule.
    Figure 11. The glcolysase protein as viewed in Cn3D, pictured similarly do Figure 4. This viewing was done by using the ball and stick model and coloring by Molecule.
      1. Is this structure a tertiary structure or quaternary structure? Explain your reasoning.
        • This is a quartenary structure as there are multiple polypeptide subunits that compose it.
      2. How many domains does DNA glycosylase have? Explain your reasoning.
        • Three. This was determined by coloring the domains (by clicking on style > coloring shortcuts > domain). Three distinct domains in the structure appear: brown, pink and blue.
      3. Can you view the same things in the structure using this program as you did when you were using Star Biochem? Why or why not?
        • Most of the same things in the structure can be viewed in Cn3D as can be viewed in Star Biochem. For example, it is easy to switch between the ball and stick model or space filling model, amongst others. In addition, Cn3D allows for easy visualization of different aspects of the entire structure by using it's coloring shortcut feature (e.g. you can view domains, elements, hydrophobicity, charge, etc.). You are also able to select residues and domains for further analysis, and zoom in/out and move the molecule as needed. StarBiochem is slightly easier to use, however, and provides greater detail in analyzing specific residues or structures within the protein.
      4. Capture at least one screenshot that is a similar a view to one of your previous screenshots as possible and post it to your page.
        • See: Figure 11.
      5. Which program do you prefer to use to view this structure? Why? You will choose to use one (or both) of these programs for your HIV Structure Project.
        • I prefer Star Biochem because it was much easier for me to use. This is likely because an instruction manual was provided, and I was forced to play around more with Cn3D. I also liked how I was able to move my mouse over specific molecules to see what amino acids were involved in different points. StarBiochem also allows greater ability to select out what features you want to analyze by being able to vary sizes of each molecule.

Weekly Assignments

Class Journals

Electronic Lab Notebook

Personal tools