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MCB 124 Protein Folding and Stability Enoch Baldwin ANSWER SET 3 page 1 I. PROTEIN STABILITY 1. Stability Measurements and Calculations First, do a van’t Hoff plot The van’t Hoff equation is lnK = - H/R(1/T) + S/R ˚C Kfold 1/T ˚K lnKf 62 2.6343601 2.985E-03 9.686E-01 64 1.38607031 2.967E-03 3.265E-01 66 0.7189229 2.950E-03 -3.300E-01 68 0.36773196 2.933E-03 -1.000E+00 We plot lnKf vs 1/T (in ˚K), fit a line to it and solve for all of the parameters in a)-c). Remember that the H and S values calculated are at the Tm only! The Van’t Hoff plot appears to be linear because the data cover a very narrow range of temperatures (if viewed over a larger temperature range, it would appear curved due to the effect of Cp) The linear equation is lnKf = 37487/T -110.92 Slope = 37487, y=intercept is -100.92 a) What is the Tm? The 1/Tm will be the X-intercept (where K=1, lnK = 0) Set for lnK = 0, solve for X (1/Tm) 1/Tm = 110.92/37487, Tm = 338˚K

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MCB 124 Protein Folding and Stability Enoch Baldwin ANSWER SET 3 page 2 b) What is the H at Tm? The slope is - H f /R or H j @ 338˚K= 37487 ˚K x -2.0 cal/mol˚K = -74,974 cal/mol = -75.0 kcal/mol c) What is S f at T m ? (Hint: don’t do a. ...) and the y=intercept is S/R so S j @ 338˚K = -110.92 x 2.0 cal/mol˚K = -222 eu Thus, H is favorable and S is unfavorable d) Using amazing biochemical tricks presently unknown to humans, you determine the cold unfolding transition. The cold T m is –41.8˚C. Calculate Cp for this protein? ( 4 pts ) Well, now you have a second Tm, where G = 0. You can use the general equation for temperature-dependence of stability G f = H f (@T 1 ) - T 2 S f (@T 1 ) + C P [ (T 2 - T 1 ) - T 2 ln(T 2 /T 1 ) ] where T 1 = 338 ˚K and T 2 = 231.2 ˚K and rearrange it to get Cp C P = (T 2 S f (@T 1 ) - H f (@T 1 ))/[ (T 2 - T 1 ) - T 2 ln(T 2 /T 1 ) ] = [(231.2 O K x -0.222 kcal/ o K-mol - -75 kcal/mol)] ÷ [(231.2 O K-338 O K) - (231.2 O K) x ln(231.2 O K/338 O K)] [23.7 kcal/mol]/(-106.8 O K-(-87.8 O K)) =[23.7 kcal/mol]/[-19.0 O K] = -1.25 kcal/mol˚K (Which is the “ideal” value)
MCB 124 Protein Folding and Stability Enoch Baldwin ANSWER SET 3 page 3 2. Name six amino acid types that you might expect to find in a protein core Ala Val Leu Ile Met Phe Cys Also: Pro Trp Tyr 2) Name six that you would more likely find on the surface. Asp Glu Lys His Arg Asn Gln Ser Thr 3) Name two that would be reasonable in either place. Gly Pro Ala Also: Tyr Cys Trp Met -these residues have some polar character and are often at partially buried positions. 3. Mutational Effects on Stability or each of the below substitutions a) predict their effects on the free energies of the folded (F) and unfolded state (U) (use "-" for a favorable change, "+" for an unfavorable change) b) for each state briefly explain why, and c) predict the overall change in stability (use same convention as for (a)) and why? 1) Surface Leu

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