Aashi Arora MCB250_Discussion2_SP24

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Feb 20, 2024

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MCB 250 Spring 2024 Discussion Worksheet - 2 Feb. 1-5 1. Write the Henderson-Hasselbach equation. pH = pKA + log([A-]/[HA]) Explain how this equation relates the concentration of the charged and uncharged forms of the side chain of aspartic acid (pKa 4) to pH. The side chain can exist in both charged and uncharged forms be where HA represents the uncharged form and A- represents the charged form. Define pH and pKa. Then, use the equation to determine the fraction of aspartic acid that is charged when: a. the pH = pKa. pH equals pKa, charged and uncharged forms are equal. b. the pH is 1 pH unit below the pKa. pH is 1 unit below pKa so the charged form is 0.1 times the concentration of the uncharged form. c. the pH is 1 pH unit above the pKa. pH is 1 unit above pKa so the charged form is 10 times the concentration of the uncharged form. d. Draw a graph of fraction charged vs. pH for the side chain of Asp. Charged vs pH for the side chain. The graph with show the sigmoidal curve and indicates a transition between the charged and uncharged forms. The midpoint of the transition corresponds to the pKa of aspartic acid. As pH deviates from the pKa, the fraction of the charged form increases or decreases exponentially.
2. The T m of a certain dsDNA is 65°C in a solution buffered at pH 7.2 and containing 0.05 M NaCl. Predict the effect of the following changes on the T m of this DNA. Explain your reasoning in each case. a. Increasing the salt concentration to 0.2 M. Increasing salt concentration will stabilize the DNA duplexes, leading to a higher Tm because higher salt concentrations shield the negatively charged phosphate groups of DNA, reducing repulsion between strands. b. Adding urea to a concentration of 40%. Urea disrupts hydrogen bonding and destabilizes DNA, leading to a lower Tm. c. Doubling the DNA concentration. Increasing DNA concentration tends to increase Tm. Higher DNA concentration provides more potential binding sites for hydrogen bonding making the duplex more stable. d. Changing the pH to 11.0. Higher pH can lead to a decrease in Tm. The increased concentration of hydroxide ions at the higher pH can disrupt hydrogen bonding In DNA, making it less table. e. Adding ethanol to the solution up to 50%. Ethanol can denature DNA and decrease Tm. Ethanol disrupts the hydrogen bonding between DNA strands, leading to decreased stability and a lower Tm. f. Replacing the NaCl with KCl at the same 0.05 M concentration. KCl is expected to have a similar stabilizing effect as NaCl so the Tm is unchanged. Both Na+ and K+ ions stabilize DNA through similar mechanisms. The change in cation type is less likely to significantly affect Tm.
3. Answer the following questions regarding the short nucleic acid shown to the right. a. Is this DNA or RNA? How do you know? DNA due to the presence of thymine instead of uracil b. Mark the 5’ and 3’ ends of the strand. 5’ end is at the top and 3’ end is at the bottom. c. Circle and name the chemical groups that mark these two ends. The Phosphate group is the 5 end and the pentose sugar at the bottom for the 3 end. d. What is the base sequence? G A T e. What is the complementary base sequence? CTA 4. Several DNA samples have been separated by agarose gel electrophoresis and stained with ethidium bromide. In the gel shown at the right, Lane 5 contains standard DNA fragments with the size (in base 400 240 160 80 bps ng/band
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pairs) and the quantity (in nanograms) for each band shown at the side. What is the approximate size and quantity of DNA in the band in Lane 3 that is marked with the arrow? At 400 5. The agarose gel at the right shows the pattern of bands obtained when a particular fragment of double-stranded DNA is digested to completion with the restriction enzymes shown. a. Which of the six restriction maps shown below is consistent with the observed pattern of restriction fragments? Not sure but maybe the third one? b. The DNA from this gel was then blotted onto a nylon filter. The filter was probed at high stringency with a radioactively labeled DNA sequence from the gene pep , which is known to lie somewhere within the region of DNA being studied here . The image to the right shows the pattern of radioactivity observed on the autoradiograph. 40 20 Autoradiograph of Southern blot filter.
Where is the pep gene located in the correct restriction map? Not sure as well but in between the A and N maybe?? 6. A microarray consists of a flat matrix with regularly placed “spots” of DNA, each with 10 7 – 10 8 copies of the same DNA sequence. You can label fragments of human genomic DNA with a fluorescent dye and hybridized the DNA probes to the microarray. If there are sequences present in the genome that can hybridize to the spot, the spot will fluoresce, which can be detected via microscopy. Indeed, you could use such an array to detect base pair differences among different individuals, so-called SNPs (single nucleotide polymorphisms). How do you imagine such a system would work? (Hint: think about the how the conditions can affect the hybridization of DNA.) Stringency conditions during hybridization are crucial. Higher stringency reduces non specific binding, allowing specific hybridization between perfectly complementary sequences. Lower stringency allows for binding even if there are some mismatches. DNA microarrays provide a high throughout method for the studying gene expression, genetic variations, and identifying SNPs in large populations. 7. a. Discuss how each of the following contribute to maintaining stable folded states of proteins: H-bonds Forming interactions between hydrogen atoms of one amino acid and the electronegative atom of another. Bonds contribute to the secondary, tertiary and quaternary structures of proteins. hydrophobic interactions Involve exclusion of water molecules from the protein’s hydrophobic core, leading to the folding of the protein to minimize exposure of hydrophobic residues to the aqueous environment.
disulfide bonds Form between two cysteine residues, creating covalent linkage. Bonds contribute to the stability of the folded state by providing a covalent link between different parts of the protein chain. van der Waals interactions Involve attractive forces between nonpolar groups that come into close proximity. These interactions contribute to the stability of the folded state by helping maintain the compact packing of hydrophobic side chains in the protein interior. b. The two major secondary structures found in proteins are -helices and -sheets. Discuss the interactions that are important for stabilizing these structures. Alpha helices are stabilized primarily by intramolecular hydrogen bonds between the oxygen of one amino acid and the amide hydrogen. The helical structure is further stabilized by van der waals interactions between adjacent side chains. Beta sheets are stabilized by hydrogen bonds formed between adjacent strands in the sheet. These can be parallel or antiparallel, and side chains extending from the strands can also contribute to the stability through van der waals interactions. c. -helices are sometimes referred to as “local structures”, while -sheets are usually not local structures. Explain. Do all proteins contain both -helices and -sheets? Alpha helices are considered local structures because they form a regular helical pattern within a short segment of the polypeptide chain. Beta sheets are considered non local structures. d. Discuss the idea of an “amphipathic helix”. An amphipathic heliz is a helical structure within a protein where one side of the helix is hydrophobic and the other side is hydrophilic.
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e. It is almost universally accepted today that the major driving force for protein folding is the hydrophobic interaction that tends to place hydrophobic side chains in the interior of the protein. Why are hydrophobic interactions more important in stabilizing the folded structures of proteins than the other forces that have been discussed? Hydrophobic interactions are considered more crucial in stabilizing folded structures compared to other forces because they drive the protein to adopt a conformation that minimizes exposure of hydrophobic residues to water. Phenomenon is a major driving force in protein folding and contributes significantly to the overall stability of the protein’s tertiary structure.

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