Bacterial proteins (Protein A, Protein G and Protein L) selectively recognise antibodies and form the basis for antibody affinity purification. The high-affinity binding between these proteins and the Fc region of antibodies often requires denaturing conditions to break, such as low pH, which limits the recovery of antibodies and their bound proteins. There is therefore a therapeutic and industrial interest in the design of antibody-binding proteins that enable milder elution conditions. To that end, "switchable" antibody-binding proteins have been designed from wild-type Protein A and Protein G. The switchable proteins that will be studied use a calcium-binding "EF-hand" motif to enable milder elution conditions by rendering the protein binding-incompetent in the absence of bound calcium. Hence, elution can be achieved by addition of chelator after binding of the antibody to the column in the presence of Ca2+ ions. EF-hands have both canonical and non-canonical forms and therefore form a diverse array of substructures. Canonical forms coordinate Ca2+ ions in a pentagonal bipyramidal coordination sphere using a flexible "EF-loop" with a length of 12 residues. Non-canonical forms can contain insertions, have deletions, or coordinate in an octahedral coordination sphere instead. In general, the EF-hand motif can accommodate this diverse array of conformations and remain binding competent. Because the EF-hand is tolerant of structural change it is an ideal target structure for directed evolution. The purpose of this project will be to probe the effects of directed evolution on the structural dynamics of the calcium-binding protein, enabling the exploration of a fitness landscape wherein molecular dynamics can be correlated with thermodynamic and kinetic data. Current data from both designed and naturally-occurring proteins suggests that the effects of calcium binding to the EF-hand are diverse. These include minor conformational changes which facilitate signal transduction in nature, to folding of the protein from a molten-globule to folded state. In addition, the conformational spaces explored by calcium-bound and apo EF-hand motifs are vast and overlapping. Since there is a great diversity in the structural responses elicited by calcium-binding, and since bound and unbound states have overlapping trajectories, molecular dynamics is proposed as an effective tool to understanding the relationship between protein sequence, fitness and the structural dynamics of binding. This computational work will be complemented by NMR spectroscopy of the same designed calcium binders. This NMR work will involve quantitative analysis of dynamics based on spin relaxation experiments, enabling determination of order parameters that will be compared to MD. This will enable the computational work to be validated against structural data obtained from experiments. The labelled material for these NMR experiments is provided by collaborators at KTH, Professor Sophia Hober lab, using a process of directed evolution with positive and negative selection pressure.