NMR Determination of Protein pK(a) Values in the Solid State

Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Journal of Physical Chemistry Letters (Impact Factor: 7.46). 05/2010; 1(10):1623-1628. DOI: 10.1021/jz1004413
Source: PubMed


Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pK(a) values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-(13)C,(15)N-labeled protein were monitored over several samples varying in pH; pK(a) values were determined from these shifts for E27, D36, and E42, and the bounds for the pK(a) of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pK(a) values give insights into the crystal packing of the protein.

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    ABSTRACT: The activity of many membrane proteins depends markedly on the pH. Pinpointing the amino acids forming the pH sensor domains of these proteins remains challenging for current experimental techniques. Combining molecular dynamics simulations and pKa predictions with in vitro transport assays, we have revealed the molecular basis of the pH dependence of the mitochondrial carrier mediating the exchanges of ADP3– and ATP4– across the inner mitochondrial membrane. We have demonstrated that the transport activity of this mitochondrial carrier depends on the protonation state of both the substrate and a unique, highly conserved residue of the protein. The original strategy proposed here offers a convenient framework for identifying pH-sensitive residues in membrane proteins in such cases where one single amino acid is involved. Our findings are envisioned to help toward the rational design of active compounds ranging from drugs to biosensors.
    Full-text · Article · Oct 2013 · Journal of Physical Chemistry Letters