Chem. 156 HW#3 Chem. 156 HW#3 3.1) In order to improve the stability of a protein, you introduce two oppositely charged amino acids into the protein by genetic engineering. The two amino acid residues you choose are about 6Å apart in the folded native structure, and they are at the surface of the protein. a) By how much (in kJ/mol) would you expect this to increase the stability of the folded protein? In other words, what would you expect for G0? You may assume that the ionic strength is low enough that it is safe to ignore screening and non-ideal effects. b) By what factor would you expect this to change the equilibrium constant for the folding process? (Make it clear whether Keq would be increased or decreased). 3.2) A molecular biologist replaces a neutral amino acid with a charged amino acid in a genetically engineered protein. Unfortunately, the side chain in question is in the interior of the folded protein (and not near any other polar groups). How much (in kJ/mol) would you expect this mutation to increase or decrease the stability of the folded protein compared to the unfolded state? [You may assume that the amino acid change has no effect on the energy of the unfolded state.] Chem. 156 HW#3 3.3) You are studying the stability of a protein by monitoring its tryptophan fluorescence while partially unfolding it in guanidinium hydrochloride (GHCl). You get the results below. a) Fill in the table below. [GHCl] 0 2.0 M 2.2 M 6M b) Fluorescence (arbitrary units) 250 225.0 211.5 200 Fraction folded 1.0 Go (KJ/mol) 0.0 --------------- --------------- What is your estimate for the stability of the protein? In other words, what is Go (in kJ/mol) for folding the protein in the absence of guanidinium? 3.4) A) What would you expect for the radius of gyration of a 100kD protein (100,000g/mol) if it is approximately spherical? [Take the density of protein to be 1.3 g/cm3]. (B) What would you expect for the radius of gyration of the same protein if it was unfolded and behaved like a semi-flexible random coil whose conformation could be modeled by a randomwalk with a step length of 20Å? [Start by figuring out what the total contour length of the chain would be, taking into account the average molecular mass of 115g/mol per amino acid along the protein chain. Use the distance of 3.7Å between consecutive C positions along the protein backbone to get the contour length].