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Download fileApproach to Study pH-Dependent Protein Association Using Constant-pH Molecular Dynamics: Application to the Dimerization of β‑Lactoglobulin
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posted on 2022-02-16, 20:44 authored by Lucie da Rocha, António M. Baptista, Sara R. R. CamposProtein–protein association
is often mediated by electrostatic
interactions and modulated by pH. However, experimental and computational
studies have often overlooked the effect of association on the protonation
state of the protein. In this work, we present a methodological approach
based on constant-pH molecular dynamics (MD), which aims to provide
a detailed description of a pH-dependent protein–protein association,
and apply it to the dimerization of β-lactoglobulin (BLG). A
selection of analyses is performed using the data generated by constant-pH
MD simulations of monomeric and dimeric forms of bovine BLG, in the
pH range 3–8. First, we estimate free energies of dimerization
using a computationally inexpensive approach based on the Wyman–Tanford
linkage theory, calculated in a new way through the use of thermodynamically
based splines. The individual free energy contribution of each titratable
site is also calculated, allowing for identification of relevant residues.
Second, the correlations between the proton occupancies of pairs of
sites are calculated (using the Pearson coefficient), and extensive
networks of correlated sites are observed at acidic pH values, sometimes
involving distant pairs. In general, strongly correlated sites are
also slow proton exchangers and contribute significantly to the pH-dependency
of the dimerization free energy. Third, we use ionic density as a
fingerprint of protein charge distribution and observe electrostatic
complementarity between the monomer faces that form the dimer interface,
more markedly at the isoionic point (where maximum dimerization occurs)
than at other pH values, which might contribute to guide the association.
Finally, the pH-dependent dimerization modes are inspected using PCA,
among other analyses, and two states are identified: a relaxed
state at pH 4–8 (with the typical alignment of the
crystallographic structure) and a compact state at
pH 3–4 (with a tighter association and rotated alignment).
This work shows that an approach based on constant-pH MD simulations
can produce rich detailed pictures of pH-dependent protein associations,
as illustrated for BLG dimerization.
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estimate free energiesthermodynamically based splinesrelaxed state compact state pearson coefficient ),observe electrostatic complementarityprotein charge distributionph molecular dynamicsmaximum dimerization occursdimerization free energydependent protein associationsdependent dimerization modesuse ionic densityrotated alignment ).inspected using pcaph md simulationsacidic ph valuesmethodological approach basedstrongly correlated sitesmd ),ph valuesprotonation stateapproach basedtypical alignmentelectrostatic interactionscorrelated sitesblg ).study phdimerization usingblg dimerizationtwo statestitratable siterelevant residuesproton occupanciesperformed usingoften overlookedoften mediatednew waymonomer facesmight contributeisoionic pointextensive networksdimeric formsdimer interfacedetailed descriptiondata generatedcrystallographic structurecontribute significantlycomputational studiesbovine blg