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

Area of Science:

• Biophysics
• Protein Chemistry
• Analytical Chemistry

Background:

• Theoretical models predict that proximity to charged molecules can alter a protein's net charge.
• This charge regulation effect is significant below the Debye length and may influence protein function without direct interaction.
• Direct measurement of charge regulation during protein crowding has been hindered by analytical challenges.

Purpose of the Study:

• To develop and apply a novel method for directly measuring protein charge regulation under conditions mimicking protein crowding.
• To investigate the impact of neighboring proteins on net protein charge without direct binding.
• To quantify changes in protein net charge due to simulated crowding.

Main Methods:

• Utilized lysine-specific protein crosslinkers (NHS ester-Staudinger pairs) to link non-interacting proteins, mimicking crowding at a defined distance (~7.9 Å).
• Employed lysine-acyl "protein charge ladders" and capillary electrophoresis to determine the net charge of monomers and regioisomeric dimers.
• Verified structural integrity and lack of direct binding using amide hydrogen/deuterium exchange and circular dichroism spectroscopy.

Main Results:

• Successfully linked myoglobin and α-lactalbumin without significant structural changes or direct binding, simulating crowding.
• Capillary electrophoretic analysis detected a small, but measurable, change in net charge (ΔZ = -0.04 ± 0.09) in the dimeric complex compared to monomers.
• The net charge of the dimer was determined to be -5.10 ± 0.07.

Conclusions:

• Demonstrated a viable method for measuring protein charge regulation under simulated crowding conditions.
• Provided the first direct experimental evidence of charge alteration in proteins due to proximity effects mimicking crowding.
• Highlighted that the magnitude of charge change is protein-specific and dependent on various factors, necessitating further investigation across different systems.