What Are We Missing by Not Measuring the Net Charge of Proteins?

Abstract

The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials—including contributions from tightly bound metal, solvent, buffer, and cosolvent ions—and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pKa. Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein—its mass—one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pKa values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔGz) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔGz contribute more to the redox potential? And what about metal binding itself? When an apo‐metalloprotein, bearing minimal net negative charge (e.g., Z=−2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool—the “protein charge ladder”—and used it to begin to answer these basic questions.

Structure and effective charge characterization of proteins by a mobility capillary electrophoresis based method

“…Proteins perform a vast number of biological functions in living organisms 1,2. Besides its primary sequence, the function of a protein greatly depends on its stereo-structure,3–5 as well as its effective charge in solution 6,7. The protein conformation and effective charge could also influence each other.…”

“…Ion CCSs22 could reflect ion structures in the gas phase,23,24 which may not have a direct relationship with their corresponding structures in solution 25. Protein charge states in the gas phase26 also have no correlation with its effective charge in solution 7. Taylor dispersion analysis (TDA) is a liquid-phase structural analysis technique, through which the diffusion coefficient and hydrodynamic radius of a particle or molecule can be measured 27.…”

Complete Charge Regulation by a Redox Enzyme Upon Single Electron Transfer

“…[10,11] For example, molecular dynamic simulations predicted that the pK a for residues in Cu(II)-Az and Cu(I)-Az (i.e., energy required for adjustments in pK a upon reduction) is G Z = 0.42 eV. [1,2] Remarkably, this G Z accounts for 46 % of the total reorganization energy measured for ET in azurin by Gray and co-workers ( T = 0.90 eV). [10,11] This current study measured charge regulation in the metalloenzyme, Cu, Zn superoxide dismutase (SOD1).…”

“…Recently, the first direct measurements of charge regulation upon single electron transfer (ET) were made in metalloproteins. [1,2] The first study examined azurin (Az), cytochrome c (Cyt c), and myoglobin (Mb). Azurin was found to partially regulate its net charge (Z) upon single ET (ΔZ ET = 0.51 ± 0.04), whereas cytochrome c did not regulate net charge, per se, but underwent a slightly larger than predicted change in net charge (ΔZ ET = 1.19 ± 0.02).…”

“…[3] Therefore, values of Z ET will be useful in rationalizing rates of ET for metalloproteins and how polypeptides tune rates of ET via electrostatic interactions between metal centers and amino acid side chains. [1,2] Charge regulation upon ET occurs from changes in the pK a of ionizable residues (or tightly bound co-factors, solvent, or buffer ions) in response to the change in metal oxidation state. [1,[4][5][6][7][8][9] Because electrostatic interactions are long range (~ 10Å in physiological buffer, but longer in the hydrophobic interior of proteins), the residues whose pK a are affected by ET can be far from the redox center.…”