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Affinity maturation increases the affinity of B-cell derived antibodies to their cognate antigens. In this study, we characterized the kinetic, structural, dynamic and thermodynamic evolution of antibodies during affinity maturation. Through single B-cell cell sorting, paired heavy and light chain sequencing, phylogenetic analysis, antibody expression, and physicochemical characterization, we were able to longitudinally analyze the stages of affinity maturation of anti-PA (B.anthracis protective antigen) antibodies. Following repeated immunizations, we observed up to an 10,000-fold increase in antibody affinity, mainly through a decrease in the off-rates. For detailed maturation analysis, we chose three antibodies lying along a single clonal branch--the clone’s unmutated common ancestor (UCA), a medium affinity antibody (MAAb) appearing after second immunization, and a high-affinity antibody (HAAb) appearing after third immunization. Most of the mutations that occur between the UCA and HAAb resulted in key changes to structural conformation. In particular, mutations change residues in the CDR-H3 region inducing the folding of the CDR-loops into a conformation that is more complementary to PA. This advantageous new antibody conformation is preserved in the unbound state, indicating that though the UCA and MAAb appear to use an induced fit and/or conformational selection mechanism, the HAAb is more rigidly lock-and-key. Thermodynamic results support this interpretation. In the first maturation step from UCA to MAAb, enthalpic improvement indicates optimization of noncovalent interactions. The second step from MAAb to HAAb predominantly involves entropic improvement by which the advantageous conformation made accessible in the first step is made more dominant via the narrowing of effectively accessible conformations, which allows better contact with PA. This is also reflected by a less significant improvement in the enthalpic component of PA-binding. Studies examining the evolving protein-dynamic characteristics further support this interpretation. In summary, we observed that a single energetic component is not responsible for increased affinity in the maturation pathways we studied. From UCA to MAAb, affinity increases through optimization of noncovalent interactions. From MAAb to HAAb, affinity increase is achieved through changes that stabilize the favorable conformation in the unbound state. A better understanding of affinity maturation can have implications for antibody engineering and vaccine development.