Almost all proteins contain charged amino acids. While the function in catalysis or binding of individual charges in the active site can often be identified, it is less clear how to assign function to charges beyond this region. Are they necessary for solubility? For reasons other than solubility? Can manipulating these charges change the properties of proteins? A combination of capillary electrophoresis (CE) and protein charge ladders makes it possible to study the roles of charged residues on the surface of proteins outside the active site. This method involves chemical modification of those residues to generate a large number of derivatives of the protein that differ in charge. CE separates those derivatives into groups with the same number of modified charged groups. By studying the influence of charge on the properties of proteins using charge ladders, it is possible to estimate the net charge and hydrodynamic radius and to infer the role of charged residues in ligand binding and protein folding.

Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.

This work describes the binding of carbonic anhydrase (CA) to mixed self-assembled monolayers (SAMs) presenting benzenesulfonamide ligands at a surface consisting primarily of tri(ethylene glycol) [(EG)3OH] groups. Surface plasmon resonance (SPR) quantified the binding of CA to the benzenesulfonamide groups. Two factors influenced the binding of CA:  (a) the density of benzenesulfonamide groups at the surface, and (b) the coverage of the surface with molecules of CA adsorbed to these benzenesulfonamide groups. At low mole-fractions of benzenesulfonamide groups in the mixed SAM where the binding of CA is highly (>90%) reversible, we observe:  (a) an approximately 10-fold decrease in the observed bimolecular rate constant for association, k on,obs, during the binding of CA (as the fraction of the surface covered by adsorbed CA increases from ∼0.15 to ∼0.35, the value of k on,obs decreases from ∼40 × 103 M-1 s-1 to ∼4 × 103 M-1 s-1); (b) almost no corresponding changes in the observed unimolecular rate constant for dissociation (k off,obs ∼0.005 s-1) during the dissociation of CA from the surface. These observations establish that k on,obs is influenced by the extent of coverage of the surface with CA, but that k off,obs is not. At low surface densities of arylsulfonamide groups, one hypothesis that rationalizes these data is that the decrease in k on,obs reflects repulsive steric interactions between molecules of CA near the surface and those already adsorbed. Each molecule of biospecifically adsorbed CA shields proximal benzenesulfonamide ligands from binding to incoming molecules of CA, and decreases the surface density of these ligands that are accessible to CA, at a rate that increases nonlinearly with the quantity of CA already adsorbed.

Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D-Ala-D-Ala (DADA)-based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins - protein charge ladders - and ACE is a physical-organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N-terminal amino group.

We present a strategy for the rapid, efficient, and accurate measurement of the coefficient of diffusion (D) of solutes using a commercial capillary electrophoresis (CE) instrument. This approach utilizes the classic analysis of Taylor of the dispersion of solutes pumped hydrostatically through glass capillaries. To obtain accurate values of D, we modified Taylor's analysis of dispersion to account for the finite time required to reach steady-state flow in the capillary when using a CE instrument. Neglecting this effect results in measured diffusivities of phenylalanine, a model solute, that are in error by as much as 60% when compared with published values. We provide an analysis of this effect and a simple strategy for avoiding these errors. Using this approach, we analyze profiles of concentration fronts and measured values of D for phenylalanine to within 5% of published values. We also analyze profiles of pulses of solute. To determine values of D accurately, measurements of dispersion first need to be made as a function of injection volume to correct for the finite width of the injection plug, before they are corrected for unsteady-state flow. This approach also yields values of D for phenylalanine to within 5% of published values. In contrast to other techniques used for the determination of D, this approach requires no fluorescent labeling and is applicable to solutes of any molecular weight.