TY - JOUR
T1 - Quantifying charge state heterogeneity for proteins with multiple ionizable residues
AU - Fossat, Martin J.
AU - Posey, Ammon E.
AU - Pappu, Rohit V.
N1 - Funding Information:
We thank Furqan Dar, Mina Farag, Andrew Lin, Kiersten Ruff, Min Kyung Shinn, and Xiangze Zeng for careful reading of the manuscript. This work was supported by grant FA9550-20-1-0241 from the Air Force Office of Scientific Research , grants R01NS089932 and 5R01NS056114 from the US National Institutes of Health , and the St. Jude Children’s Research Collaborative on Membraneless Organelles .
Funding Information:
We thank Furqan Dar, Mina Farag, Andrew Lin, Kiersten Ruff, Min Kyung Shinn, and Xiangze Zeng for careful reading of the manuscript. This work was supported by grant FA9550-20-1-0241 from the Air Force Office of Scientific Research, grants R01NS089932 and 5R01NS056114 from the US National Institutes of Health, and the St. Jude Children's Research Collaborative on Membraneless Organelles.
Publisher Copyright:
© 2021 Biophysical Society
PY - 2021/12/21
Y1 - 2021/12/21
N2 - Ionizable residues can release and take up protons and this has an influence on protein structure and function. The extent of protonation is linked to the overall pH of the solution and the local environments of ionizable residues. Binding or unbinding of a single proton generates a distinct charge microstate defined by a specific pattern of charges. Accordingly, the overall partition function is a sum over all charge microstates and Boltzmann weights of all conformations associated with each of the charge microstates. This ensemble-of-ensembles description recast as a q-canonical ensemble allows us to analyze and interpret potentiometric titrations that provide information regarding net charge as a function of pH. In the q-canonical ensemble, charge microstates are grouped into mesostates where each mesostate is a collection of microstates of the same net charge. Here, we show that leveraging the structure of the q-canonical ensemble allows us to decouple contributions of net proton binding and release from proton arrangement and conformational considerations. Through application of the q-canonical formalism to analyze potentiometric measurements of net charge in proteins with repetitive patterns of Lys and Glu residues, we determine the underlying mesostate pKa values and, more importantly, we estimate relative mesostate populations as a function of pH. This is a strength of using the q-canonical approach that cannot be replicated using purely site-specific analyses. Overall, our work shows how measurements of charge equilibria, decoupled from measurements of conformational equilibria, and analyzed using the framework of the q-canonical ensemble, provide protein-specific quantitative descriptions of pH-dependent populations of mesostates. This method is of direct relevance for measuring and understanding how different charge states contribute to conformational, binding, and phase equilibria of proteins.
AB - Ionizable residues can release and take up protons and this has an influence on protein structure and function. The extent of protonation is linked to the overall pH of the solution and the local environments of ionizable residues. Binding or unbinding of a single proton generates a distinct charge microstate defined by a specific pattern of charges. Accordingly, the overall partition function is a sum over all charge microstates and Boltzmann weights of all conformations associated with each of the charge microstates. This ensemble-of-ensembles description recast as a q-canonical ensemble allows us to analyze and interpret potentiometric titrations that provide information regarding net charge as a function of pH. In the q-canonical ensemble, charge microstates are grouped into mesostates where each mesostate is a collection of microstates of the same net charge. Here, we show that leveraging the structure of the q-canonical ensemble allows us to decouple contributions of net proton binding and release from proton arrangement and conformational considerations. Through application of the q-canonical formalism to analyze potentiometric measurements of net charge in proteins with repetitive patterns of Lys and Glu residues, we determine the underlying mesostate pKa values and, more importantly, we estimate relative mesostate populations as a function of pH. This is a strength of using the q-canonical approach that cannot be replicated using purely site-specific analyses. Overall, our work shows how measurements of charge equilibria, decoupled from measurements of conformational equilibria, and analyzed using the framework of the q-canonical ensemble, provide protein-specific quantitative descriptions of pH-dependent populations of mesostates. This method is of direct relevance for measuring and understanding how different charge states contribute to conformational, binding, and phase equilibria of proteins.
UR - http://www.scopus.com/inward/record.url?scp=85120777466&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2021.11.2886
DO - 10.1016/j.bpj.2021.11.2886
M3 - Article
C2 - 34826385
AN - SCOPUS:85120777466
SN - 0006-3495
VL - 120
SP - 5438
EP - 5453
JO - Biophysical Journal
JF - Biophysical Journal
IS - 24
ER -