During the affinity maturation process the immune system produces antibodies with higher specificity and activity through various rounds of somatic hypermutations in response to an antigen. Elucidating the affinity maturation process is fundamental in understanding immunity and in the development of biotherapeutics. Therefore, we analyzed 10 pairs of antibody fragments differing in their specificity and in distinct stages of affinity maturation using metadynamics in combination with molecular dynamics (MD) simulations. We investigated differences in flexibility of the CDR-H3 loop and global changes in plasticity upon affinity maturation. Among all antibody pairs we observed a substantial rigidification in flexibility and plasticity reflected in a substantial decrease of conformational diversity. To visualize and characterize these findings we used Markovstates models to reconstruct the kinetics of CDR-H3 loop dynamics and for the first time provide a method to define and localize surface plasticity upon affinity maturation.
Antibodies have emerged as one of the fastest growing classes of biotherapeutic proteins. To improve the rational design of antibodies, we investigate the conformational diversity of 16 different germline combinations, which are composed of 4 different kappa light chains paired with 4 different heavy chains. In this study, we systematically show that different heavy and light chain pairings strongly influence the paratope, interdomain interaction patterns and the relative VH-VL interface orientations. We observe changes in conformational diversity and substantial population shifts of the complementarity determining region (CDR) loops, resulting in distinct dominant solution structures and differently favored canonical structures. Additionally, we identify conformational changes in the structural diversity of the CDR-H3 loop upon different heavy and light chain pairings, as well as upon changes in sequence and structure of the neighboring CDR loops, despite having an identical CDR-H3 loop amino acid sequence. These results can also be transferred to all CDR loops and to the relative VH-VL orientation, as certain paratope states favor distinct interface angle distributions. Furthermore, we directly compare the timescales of sidechain rearrangements with the well-described transition kinetics of conformational changes in the backbone of the CDR loops. We show that sidechain flexibilities are strongly affected by distinct heavy and light chain pairings and decipher germline-specific structural features co-determining stability. These findings reveal that all CDR loops are strongly correlated and that distinct heavy and light chain pairings can result in different paratope states in solution, defined by a characteristic combination of CDR loop conformations and VH-VL interface orientations. Thus, these results have broad implications in the field of antibody engineering, as they clearly show the importance of considering paired heavy and light chains to understand the antibody binding site, which is one of the key aspects in the design of therapeutics.
The combination of several active substances into one carrier is often limited due to solubility, stability and phase-separation issues. These issues have been addressed by an innovative capsule design, in which nanocapsules are assembled on the microcapsule surface by electrostatic forces to form a pH-responsive hierarchical capsule@capsule system. Here, melamine-formaldehyde (MF) microcapsules with a negative surface charge were synthesized and coated with a novel MF-polyethyleneimine (PEI) copolymer to achieve a positive charge of ζ = + 28 mV. This novel coating procedure allows the electrostatic assembly of negatively charged polyl-lactide (PLLA, ζ = À 19 mV) and poly-(lactide-co-glycolide) (PLGA, ζ = À 56 mV) nanocapsules on the microcapsule surface. Assembly studies at pH 7 gave a partial surface coverage of PLLA nanocapsules and full surface coverage for PLGA nanocapsules. The pH-responsive adsorption and desorption of nanocapsules was shown at pH 7 and pH 3.
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