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MCB124_ANSWERS_SET2 - MCB 124 Enoch Baldwin Protein...

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MCB 124 Protein Quaternary Structure, Nucleic Acid Structure Enoch Baldwin PROBLEM SET II page 1 I. Quaternary Structure A. You are a Junior Researcher in Dr. Rico Mendation’s Lab. For your first project you are given a human protein from blood, GETNA, that has the following properties. 1) Using SDS/PAGE (denaturing electrophoresis), you see 2 bands of apparent molecular weight of ~30 kD and ~10 kD. You call these GETNA-1 and GETNA-2. 2) A partial peptide sequence allows you to identify the two cDNAs (cDNAs correspond to the spliced mRNAs, that is they contain no exons, only coding sequence), which each show a single reading frame. For GETNA-1, the reading frame is 885 bases, while for GETNA-2, the reading frame is 273 bases. The sequence of GETNA-1 implies that it is an ATPase (binds and hydrolyzes ATP). 3) Gel filtration chromatography, ultracentrifugation, and light scattering give similar results that depend on salt concentration and the presence/absense of ATP. a) In 150 mM NaCl/10 mM ATP, a single species of apparent molecular weight of 120 kD is observed. b) in 150 mM/2 mM ATP, two species are observed 120 kD and 40 kD. c) in 150 mM NaCl/noATP only a 40 kD species is observed. d) at 500 mM/20 mM ATP four species are observed, 120 kD, 40 kD, 30 kD and 10 kD. e) 500 mM NaCl/no ATP only two species are observed, 30 kD and 10 kD. This protein is trimer of heterodimers (GETNA-1/GETNA-2) 3 in the presence of ATP and physiological salt (120 kD = 3 x (30 kD + 10 kD)). The trimer dissociates into the heterodimers in the absence of ATP, in other words, ATP promotes trimerization. High salt further dissociates the heterodimer, presumably by disruption ionic intersubunit interactions. ATP can stabilize the heterodimers in the trimer against salt-induced dissociation by Le Chatlier’s principle. Salt +ATP GETNA-1 + GETNA-2  GETNA-1/GETNA-2  (GETNA-1/GETNA-2) 3  ATP complex The subunit weights are confirmed by their sequences, the general rule of thumb being the each residue averages 110 Da. The number of protein residues is for: GETNA-1 = (885 bp/3bp/codon) x 110 Da/residue = 32.4 kD, GETNA-2 = (273/3 x 110) = 10.0 kD. The weights are no exactly those from the hydrodynamic measurements because the measurements are not that accurate. B. Because of your brilliance, you are given another protein to characterize, an ATPase, GOTNF, this time from whole white cells. 1) By SDS/PAGE and Gel filtration (in the absence of ATP) you get the same molecular weight, 20 kD. This is the monomer without ATP bound 2) If you fail to use DNaseI in the prep (an enzyme that is used to degrade DNA in tissue preparations), the protein is insoluble. GOTNF binds to the chromosomal DNA which spins down with the pellet because it is very large (in chromosomes).
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MCB 124 Protein Quaternary Structure, Nucleic Acid Structure Enoch Baldwin PROBLEM SET II page 2 3) In the presence of ATP, Gel filtration of pure GOTNF (sorry, forgot that) gives multiple peaks of 20, 40, 60, 80, and 100 kD with the rest in the void volume (> 300 kD).
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