Proteolytic footprinting titrations for estimating ligand-binding constants and detecting pathways of conformational switching of calmodulin.
To dissect the chemical basis for interactions controlling regulatory properties of macromolecular assemblies, it is essential to explore experimentally the linkage between ligand binding, conformational change, and subunit assembly. There are many advantages to using techniques that will probe the occupancy of individual binding sites or monitor conformational responses of individual residues, as described here. Proteolytic footprinting titrations may be used to infer binding free energies for ligands interacting with multiple sites or domains and to detect otherwise unrecognized "silent" interdomain interactions. Microgram quantities of pure protein are required, which is low relative to the hundreds of milligrams needed for comparable discontinuous equilibirum titrations monitored by NMR. By running comparative studies with several proteases, it is easy to determine whether resulting titration curves are consistent, independent of the protease used and therefore representative of the structural response of the protein to ligand binding or other differences in solution conditions (pH, salt, temperature). The results from multiple techniques (e.g., NMR, fluorescence, and footprinting) applied to aliquots from the same discontinuous titration may be compared easily to test for consistency. Classic methods for determining thermodynamic and kinetic properties of calcium binding to calmodulin include filter binding and equilibrium or flow dialysis (employing the isotope 45Ca), spectroscopic studies of stopped-flow fluorescence, calorimetry, and direct ion titrations. A cautionary note is that many different sets of microscopic data would be consistent with a single set of macroscopic constants determined by classic methods. This was well illustrated in Fig. 9. Thus, while it is important to compare results with those obtained by classic binding methods, they are, by definition, incapable of resolving the microscopic constants of interest. Thus, there is only one "direction" for comparison. Quantitative proteolytic footprinting titrations applied to studying calmodulin provided the first direct quantitative estimate of negative interactions between domains. Although studies of site-knockout mutants had suggested interactions between domains, this approach gave the first evidence for the pathway of anticooperative interactions between domains by showing that helix B responds structurally to calcium binding to sites III and IV in the C-domain. Despite two decades of study of calmodulin and the application of limited proteolysis studies to the apo and fully saturated forms, this finding emerged only when titration studies were undertaken as described. This highlights the general observation that while the behavior of the intermediate states in a cooperative switch are the key elements of the transition mechanism, they are the most difficult to observe. The unexpected finding that the isolated domains are nearly equivalent in their calcium-binding properties (Fig. 23 B) leaves us with many of the questions we had at the start: How does the sum of two nearly equivalent domains result in a molecule that switches sequentially rather than simultaneously? But it underscores why it is not yet possible to understand similar proteins by sequence gazing alone.
Shea
M A
MA
Department of Biochemistry, University of Iowa, College of Medicine, Iowa City 52242-1109, USA.
Sorensen
B R
BR
Pedigo
S
S
Verhoeven
A S
AS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013917:Thrombin / Q000378:metabolism
D002147:Calmodulin; D008024:Ligands; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013917:Thrombin; D002118:Calcium