Measuring how two proteins affect each other's net charge in a crowded environment

• Published in: Protein science Vol. 30; no. 8; pp. 1594 - 1605
• Main Authors: Dashnaw, Chad M., Koone, Jordan C., Abdolvahabi, Alireza, Shaw, Bryan F.
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.08.2021; Wiley Subscription Services, Inc
• Subjects: Binding; Capillary electrophoresis; charge regulation; chemical crosslinking; Circular dichroism; Cross-Linking Reagents - chemistry; Crosslinking; Crowding; Debye length; Deuterium; Dichroism; Dimers; Electrophoresis; Electrophoresis, Capillary; Full‐Length Paper; Full‐Length Papers; Hydrogen-deuterium exchange; Lactalbumin; Lactalbumin - chemistry; Ladders; Lysine; macromolecular crowding; Mimicry; Monomers; Myoglobin - chemistry; Myoglobins; Protein Conformation; protein electrostatics; Protein Folding; Protein structure; Proteins; Proteins - chemistry; Spectroscopy; Static Electricity; charge regulation; macromolecular crowding; chemical crosslinking; capillary electrophoresis; protein electrostatics
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.4092

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 (Zmonomer = −0.43 ± 0.01) and α‐lactalbumin (Zmonomer = −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 ΔZ = −0.04 ± 0.09 upon crowding by this pair (Zdimer = −5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.