Quiz 11: CH 8
The binding of protein A to two other proteins, B and C, has been studied with fluorescence anisotropy, and the results of the measurements have been plotted in the following schematic diagram. Given equal kon rate constants, which complex do you think has a longer half-life? What is the value of K¬a (the association constant) for this complex? (Please refer to the image in the "MCB_BIOL407 Quiz 11 Figures" Question #5) a) A-B; 10^5 L/mol b) A-B; 10^-5 L/mol c) A-C; 1.5 × 10^5 L/mol d) A-C; 6.7 × 10^4 L/mol e) A-C; 6.7 × 10^-4 L/mol
a) A-B; 10^5 L/mol feedback: The A-B complex formation has a lower Kd (10 µM) as shown by the curves. Accordingly, it should have a lower koff (given identical kon values), and therefore a longer half-life. The Ka is the inverse of Kd.
In purifying proteins by column chromatography, elution can be performed in different ways, depending on the type of matrix. For example, bound proteins can be eluted by gradually increasing or decreasing salt concentration in the solution applied to the column. For which of the following matrices do you think increasing and decreasing salt concentration, respectively, are used for elution? a) Ion-exchange; hydrophobic b) Affinity; ion-exchange c) Hydrophobic; affinity d) Hydrophobic; ion-exchange e) Ion-exchange; gel-filtration
a) Ion-exchange; hydrophobic feedback: Increasing salt concentration would weaken the charge-charge interactions between bound proteins and an ion-exchange matrix. Decreasing salt concentration, on the other hand, weakens the hydrophobic interactions between bound proteins and a hydrophobic matrix.
In sequence alignments such as those generated by a BLAST search, the significance of the alignment can be presented as an E-value, which specifies how likely it is for a match this good to be found in a database of a given size by chance. You have scanned a large sequence database with a sequence query, hoping to find a significant match. Which one would you look at based on the E value below? a) 1 b) 0 c) 1 x 10^-200 d) 100 e) none of the above
b) 0 feedback: A smaller E¬-value represents a more significant match. Zero would mean an exact match.
How is tandem affinity purification distinct from other purification schemes such as simple affinity or ion-exchange chromatography? a) In its use of antibodies b) In its use of proteases c) In its use of glutathione S-transferase d) In its use of viral peptides e) In its use of limited washing steps
b) In its use of proteases feedback: Since tandem affinity purification (TAP)-tagging relies on proteolytic cleavage for elution of the protein from the first column, the protein can be washed extensively at this step to remove most contaminants. Additionally, it can be purified further due to the presence of another, non-cleaved, tag.
To create cellular factories for monoclonal antibody production, cells derived from an immortalized B lymphocyte cell line (M) are cultured together with antibody-secreting B lymphocytes (B) on a so-called HAT medium. The medium contains an inhibitor of DHFR, an enzyme essential for de novo nucleotide biosynthesis. Additionally, it contains substrates for the only alternative nucleotide biosynthetic pathway called the salvage pathway. The enzyme HGPRT is essential for the salvage pathway. One of the cells mentioned above lacks the gene encoding one of the enzymes mentioned. Considering the role of the HAT medium in selecting for hybrid cells that are both immortalized and secrete antibodies, which cell (B or M) do you think is missing an enzyme? Which enzyme is missing in this cell? a) M- DHRF b) M-HGPRT c) B-DHFR d) B-HGPRT
b) M-HGPRT feedback: The transformed myeloma (M) cells are immortalized but are not selected for in HAT media. This is because they lack HGPRT and their DHFR-dependent pathway is also inhibited. Non-fused B cells can still make their nucleotides by the salvage pathway but can only proliferate for a few generations. The hybrid cells, on the other hand, are both immortalized and capable of carrying out the salvage pathway.
The EcoRI restriction enzyme recognizes and cuts at the sequence GAATTC. Such an enzyme is expected to cut a typical double-stranded DNA at 1 in every ... sites. a) about 400 b) about 4,000 c) about 40,000 d) about 400,000 e) about 4 million
b) about 4,000 feedback: Such a restriction site is expected to occur by chance in 1 in 46 random hexamers.
Your friend has cut a 3000-nucleotide-pair circular plasmid with either or both restriction enzymes A and B, and has separated the digestion products by electrophoresis. The results are shown in the schematic drawing below, with the approximate size of the fragments indicated above each band. Based on these results, what is the closest distance (in nucleotide pairs) between an A restriction site and a B restriction site on this plasmid? (Please refer to the image in the "MCB_BIOL407 Quiz 11 Figures" Question #10) a) 400 b) 600 c) 1000 d) 1200 e) 1600
c) 1000 feedback: One of the B sites is 1000 nucleotide pairs away from the A site, and the second B site is another 400 nucleotide pairs further, making it 1400 nucleotide pairs away in one direction and 1600 nucleotide pairs away in the other direction around the circular plasmid.
Which mass spectrometry instrument is most suitable for determining all the proteins present in an organelle? a) MALDI-TOF b) MS/MS c) LC-MS/MS d) GC-MS
c) LC-MS/MS feedback: In this set-up, a chromatographic separation (liquid chromatography; LC) precedes the feeding of different peptides or proteins derived from the organelle into the tandem mass spectrometer (MS) system.
Migration of proteins during separation by SDS polyacrylamide-gel electrophoresis (SDS-PAGE) is generally related to the logarithm of their molecular weight, as depicted in the qualitative graph below. If a protein mixture containing proteins of evenly distributed molecular weights (e.g. 10, 20, 30, 40, etc.) is separated by SDS-PAGE, which of the following staining patterns would you expect to observe? (Please refer to the image in the "MCB_BIOL407 Quiz 11 Figures" Question #2) a) Lane A pattern b) Lane B pattern c) Lane C pattern d) Lane D pattern
c) Lane C pattern feedback: Within the linear range shown in the graph, smaller proteins are better resolved (separated) in SDS-PAGE compared to larger proteins.
You have carried out a genetic screen in a species of fish, and have identified a new gene in this organism. You have cloned and sequenced the gene, but do not know anything about the biochemical function of the gene product. Which of the following is the most sensible next step in order to get clues about the function? a) Purifying the protein product and determining its structure by x-ray diffraction b) Screening for small molecules that perturb gene function c) Using the BLAST algorithm to scan sequence databases for similar sequences d) Using MS/MS to sequence the gene product without the need for purification e) Mutating the gene and performing reverse genetics
c) Using the BLAST algorithm to scan sequence databases for similar sequences feedback: Searching a collection of known gene and protein sequences is typically done over the Internet using fast alignment algorithms such as BLAST. The clues generated from such searches typically help the investigator in future, direct experimental studies.
Where do individual peptides get fragmented in a tandem mass spectrometry (MS/MS) set-up? a) electron ion source b) mass filter c) inert gas chamber d) mass analyzer e) detector
c) inert gas chamber feedback: In MS/MS, a collision chamber containing a high-energy inert gas connects the two mass analyzers (the first one acting as a mass filter) and is the place where fragmentation of precursor ions takes place.
Which of the following chromatography methods resembles immunoprecipitation in the purification of proteins from mixtures? a) Ion-exchange chromatography b) Gel-filtration chromatography c) Hydrophobic interaction chromatography d) Affinity chromatography
d) Affinity chromatography feedback: Immunoprecipitation is a variation on the theme of affinity chromatography.
The AgeI restriction enzyme recognizes the sequence ACCGGT and cuts each strand between the first (A) and the second (C) nucleotide. This can be represented as A|CCGGT, with the vertical bar indicating the cleavage position. If a DNA fragment is cut with AgeI at both ends, cutting a plasmid with which of the following enzymes would then allow insertion of the fragment into the plasmid by simple ligation of sticky ends? a) FseI, with the recognition sequence GGCCGG|CC b) AatI, with the recognition sequence AGG|CCT c) BstZI, with the recognition sequence C|GGCCG d) MroI, with the recognition sequence T|CCGGA e) None of the above
d) MroI, with the recognition sequence T|CCGGA feedback: Both AgeI and MroI generate the same sticky end (with the sequence CCGG) as a 5′ overhang after cleavage.
In SDS-PAGE of proteins, ... a) the proteins are unfolded while being separated. b) the proteins are separated by size, mostly independent of their native charge. c) large proteins move more slowly. d) an ionic detergent is used. e) All of the above.
e) All of the above. feedback: SDS (sodium dodecyl sulfate) is a negatively charged detergent that can release proteins from their binding partners and unfold them into extended polypeptide chains. It also masks each protein's intrinsic charge. Consequently, during SDS polyacrylamide-gel electrophoresis (SDS-PAGE), proteins are separated mainly based on their size, and not their shape or intrinsic charge; larger proteins move more slowly because the gel matrix hinders their migration to a higher extent compared to smaller proteins.
