Learning Goal: Calculation of the apparent denaturation constant Kapp for the sequence of equilibria N i , D Assume that an observed physical property is given by y = fNyN + fDyD + fiyi where fN, fD, andfi are the fractions of structured (N), unstructured (D) and intermediately structured (i) protein and yN, yD, andy; are the physical property values associated with the N, D, and i protein forms. If a single intermediate is present then fN + fD+ fi =1 Part A The apparent fractional denaturation is defined as fapp = y-yN yD yN = fD + fidi. Note only fapp is measureable and will not equal fo if intermediates are present and depending on the value of the difference parameter di = yi-yN yD yN Assume fN = 0.60 and fi = 0.10 and yi = yN so that property y for the intermediate and the N form are identical. Calculate fapp IVO AEQ ? fapp = Submit Request AnswerPart B Assume fN = 0.60 and fi = 0.10 and yi = yo so that property y for the intermediate and the D form are identical. Calculate fapp AED ? fapp = Submit Request Answer Part C Based on your calculations in parts A and B, which of the following statements is correct? O As yi approaches yN, fappapproachesfN O As yi approaches yD, fappapproaches fo O As yi approaches yN, fappapproaches fo Submit Request AnswerPart D V Assume the N form , the intermediate i, and the D form are in a sequence of equiibria N i , D. We define the equilibrium constants Ki = i and KD = JD IN Using the conditions in part B calculate Ki and KD Ki, KD = 0.167,0.500 Submit Previous Answers V Correct Part E V The apparent folding equilibrium constant is given by Kapp = (1-fapp Japp . Jing this definition and the equations fN + fD + fi = 1 and fapp = fD + fidi, given values of Ki and KD , Kapp is a function of di: Kapp(di) = KD(1+diKi/KD) (1+(1-di) Ki) Kapp is the only equilibrium constant that can be measured from this system of folding equilibria N i - D. Clearly Kapp = KD if there is no intermediate. But if intermediate is present how does Kapp vary as a function of di? Assuming fN = 0.600 and fi = 0.100 calculate values of Kapp for di = 0 di = 0.5 and di = 1 Kapp(0), Kapp(0.5), Kapp(1) = 0.428,0.539,0.667 Submit Previous AnswersV Part F Assuming fD = 0.6 and fi = 0.1 for what value of di does Kapp = KD? AED d ? di = 0.667 Submit Previous Answers Request Answer X Incorrect; Try Again; 5 attempts remaining