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

Abstract

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 = À0.43 ± 0.01) and α-lactalbumin (Z monomer = À4.63 ± 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 = À0.04 ± 0.09 upon crowding by this pair (Z dimer = À5.10 ± 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.

A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor

“…The results of nonselective acylation are supported by previous studies showing that small molecule acylating agents (e.g., anhydrides, N‐hydroxysuccinimide [NHS]‐esters, and aryl esters) acylate lysine in proteins semi‐randomly 47,64–66 . Even partially buried lysine residues are reactive with small organic molecules, because these lysines can associate with amphiphilic/hydrophobic molecules (in some cases, more so than solvent accessible lysine residues) 67 .…”

“…This issue—the magnitude by which two proteins affect each other's net charge in a crowded environment—is poorly understood 57–59 . The question has been examined with Monte Carlo simulations 60 and protein charge ladders 47 . Lund and Jonsson simulated how the net charge of calbindin and lysozyme change as they approach each other at a distance of ~25 Å (mass center to mass center) and ionic strength of I = 0.006 M (pH 4.0).…”

“…45,46 This current study expands upon a recently reported method of semi-random chemical crosslinking to mimic the crowding of proteins by other proteins. 47 Although crosslinking is widely used to trap specific proteinprotein interactions, [48][49][50][51] we found that commercial crosslinkers can link noninteracting proteins (of various net charge) in a quasi-random fashion. 47 This semirandom crosslinking method allows us to eliminate possible site-specific electrostatic effects that might arise from a genetically engineered fusion protein.…”

“…This net charge is nearly identical to Z formal = +13.09 ± 0.04, that is, the sum of the net charges of RNase A (Figure 2a, Z CE = +5.94 ± 0.02) and Cyt c monomers (Figure 2b, Z CE = +8.50 ± 0.04), when correcting for Staudinger crosslinking at two lysine (previously measured to be ΔZ = À1.34). 47 Mass spectrometry (MS) was used to assess the extent of modification of lysine in each protein with its bifunctional linker before mixing and crosslinking (Figure S2; Az = 83 Da, PPh 3 = 346 Da). Our goal was to only attach between 1 and 3 linkers to each protein.…”

“…Using protein "charge ladders" and chemical crosslinking, we recently found that this type of charge regulation was small for a particular pair of proteins (Mb and α-lactalbumin) at I = 0.025 M. 47 However, larger effects might be observed at lower ionic strength (e.g., at I < 0.01 M), which might be physiologically relevant in membrane interiors or during liquid-liquid phase separation. 62,63 Nevertheless, how any of these changes in pK a 's of ionizable residues-large or small-might impact enzymatic activity in vitro has not been measured or investigated (to our knowledge).…”