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Measuring how two proteins affect each other's net charge in a crowded environment
Author(s) -
Dashnaw Chad M.,
Koone Jordan C.,
Abdolvahabi Alireza,
Shaw Bryan F.
Publication year - 2021
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.4092
Subject(s) - myoglobin , chemistry , macromolecular crowding , charge (physics) , circular dichroism , crystallography , charge density , protein structure , hydrogen–deuterium exchange , electrophoresis , biophysics , mass spectrometry , macromolecule , biochemistry , chromatography , biology , physics , quantum mechanics
Theory predicts that the net charge ( Z ) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester‐Staudinger pairs) can be used to mimic crowding by linking two non‐interacting proteins at a maximal distance of ~7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine‐acyl “protein charge ladders” and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin ( Z monomer = −0.43 ± 0.01) and α‐lactalbumin ( Z monomer = −4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of Δ Z = −0.04 ± 0.09 upon crowding by this pair ( Z dimer = −5.10 ± 0.07). These small values of Δ Z are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.