Effect of peptide binding on amide proton exchange rates in the PDZ2 domain from human phosphatase hPTP1E

Amide hydrogen-deuterium exchange rates were measured in the PDZ2 domain fr om human phosphatase hPTP1E by H-1-N-15 heteronuclear NMR spectroscopy. Pro tection factors were calculated for the slowly exchanging hydrogens in both the free PDZ2 domain and its complex with an octapeptide peptide, R-N-E-I- Q-S-L-V, derived from the C-terminus of the Fas receptor. Aside from a shor t ex-helical region al (amino acids A-45 to D-49), the pattern of highly pr otected amides correlated well with the presence of hydrogen bonds in eleme nts of the secondary structure. Hydrogen-bonded amides showed relatively fa st exchange rates with half-lives of less than 9 h at pD 7.6 and 8 degrees C. Protection factors, calculated as the ratio of theoretical (denatured) a nd observed exchange rates, showed less dispersion in maximal values than d id the actual exchange rates. This behavior and the large pH dependence of the exchange rates suggest that amide exchange is close to the EX2 limit. I n this limit, exchange of the most protected amides occurs through a global unfolding mechanism. The free energy of the unfolding calculated from the largest protection factors is 4.8 +/- 0.4 kcal/mol (1 cal = 4.184 J). This Delta G degrees closely matches the value measured by experiments with guan idine hydrochloride and fluorescence emission spectroscopy. Peptide binding to PDZ2 resulted in mostly global effects and stabilized the folded domain by 1.4 kcal/mol.