pH-Dependent Conformational Changes in Proteins and Their Effect on Experimental pK(a)s: The Case of Nitrophorin 4

NATALI DIRUSSO; DARIO A ESTRIN; MARCELO A. MARTI; ADRIAN E ROIRBERG

PLOS COMPUTATIONAL BIOLOGY

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2012 p. 100276 - 100276

1553-734X

The acid-base behavior of amino acids is an important subject of study due to their prominent role in enzyme catalysis,substrate binding and protein structure. Due to interactions with the protein environment, their pKas can be shifted fromtheir solution values and, if a protein has two stable conformations, it is possible for a residue to have different??microscopic??, conformation-dependent pKa values. In those cases, interpretation of experimental measurements of the pKais complicated by the coupling between pH, protonation state and protein conformation. We explored these issues usingNitrophorin 4 (NP4), a protein that releases NO in a pH sensitive manner. At pH 5.5 NP4 is in a closed conformation whereNO is tightly bound, while at pH 7.5 Asp30 becomes deprotonated, causing the conformation to change to an open statefrom which NO can easily escape. Using constant pH molecular dynamics we found two distinct microscopic Asp30 pKas: 8.5in the closed structure and 4.3 in the open structure. Using a four-state model, we then related the obtained microscopicvalues to the experimentally observed ??apparent?? pKa, obtaining a value of 6.5, in excellent agreement with experimentaldata. This value must be interpreted as the pH at which the closed to open population transition takes place. Moregenerally, our results show that it is possible to relate microscopic structure dependent pKa values to experimentallyobserved ensemble dependent apparent pKas and that the insight gained in the relatively simple case of NP4 can be usefulin several more complex cases involving a pH dependent transition, of great biochemical interest.