To address how long-range electrostatic force can affect antibody-antigen binding, we focused on the interactions between human cardiac troponin I and its specific single-chain antibodies (scFvs). We first isolated two scFvs against two linear epitopes with distinct isoelectric points. For the scFv against the acidic epitope (A1scFv), we mutated five residues of framework region 3 of the light chain to Lys or Arg, designated as the K- or R-mutant, respectively. For the scFv against the basic epitope (A2scFv), we mutated four or three residues in framework region 3 of the light or heavy chain to Asp, to generate the VL- and VH-mutant, respectively. Surface plasmon resonance analyses showed that the kon values of all of the mutants were greater than that of wild type, even though framework region 3 does not make direct contact with the epitope. The affinity of the K-mutant was pM range, and that of the R-mutant improved further by more than two orders of magnitude due to a decrease in the dissociation rate constant. For the A2scFv mutants, the affinity of the VL-mutant for its target improved through an increase in the kon value without a decrease in the koff value. The stability slightly decreased in all mutants. These results suggest that introducing electrostatic interaction can improve the affinity of an antibody for its target, even if the mutation reduces stability of the antibody.
Submitter: Marie Ary
Submission Date: Nov. 8, 2018, 6:03 p.m.
|Number of data points||33|
|Proteins||A2scFv (anti-cardiac troponin I scFv) ; A1scFv (anti-cardiac troponin I scFv)|
|Assays/Quantities/Protocols||Experimental Assay: Tm ; Experimental Assay: Kd ; Experimental Assay: k_off ; Experimental Assay: k_on|
|Libraries||Kd, k_on, k_off, Tm for A2scFv-troponin I interactions (Table I) ; Kd, k_on, k_off, Tm for A1scFv-troponin I interactions (Table I)|
|Structure ID||Release Date||Resolution||Structure Title|
|2DQT||2006-05-30T00:00:00+0000||1.8||High resolution crystal structure of the complex of the hydrolytic antibody Fab 6D9 and a transition-state analog|
|2DQU||2006-05-30T00:00:00+0000||1.7||Crystal form II: high resolution crystal structure of the complex of the hydrolytic antibody Fab 6D9 and a transition-state analog|
|2DTM||2006-07-13T00:00:00+0000||2.25||Thermodynamic and structural analyses of hydrolytic mechanism by catalytic antibodies|
|3AB0||2009-11-28T00:00:00+0000||3.09||Crystal structure of complex of the Bacillus anthracis major spore surface protein BclA with ScFv antibody fragment|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|90.9||A1scFv (anti-cardiac troponin I scFv)||P18527||HVM56_MOUSE|