ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K b ) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T m can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30°C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T m . The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using 15N and 2H NMR relaxation methods. NMR relaxation studies of the complex were carried out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-bearing side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains.

Magnetic relaxation has been used extensively to study and characterize biological tissues. In particular, spin-lattice relaxation in the rotating frame (T 1) of water in protein solutions has been demonstrated to be sensitive to macromolecular weight and composition. However, the nature of the contribution from low frequency processes to water relaxation remains unclear. We have examined this problem by studying the water T1 dispersion in peptide solutions ( 14 N-and 15 N-labeled), glycosaminoglycan solutions, and samples of bovine articular cartilage before and after proteoglycan degradation. We find in model systems and tissue that hydrogen exchange from NH and OH groups to water dominates the low frequency water T 1 dispersion, in the context of the model used to interpret the relaxation data. Further, low frequency dispersion changes are correlated with loss of proteoglycan from the extra-cellular matrix of articular cartilage. This finding has significance for the noninvasive detection of matrix degradation.rotating frame ͉ T1 ͉ extra-cellular matrix ͉ osteoarthritis P rotein degradation with a loss of proteoglycan (PG) from the extra-cellular matrix is thought to be an initiating event of early osteoarthritis (1). A noninvasive imaging method that can monitor the progression of the disease would be highly desirable for the longitudinal evaluation of disease progression and the utility of therapeutic interventions. Because of the excellent soft tissue contrast and its noninvasive nature, MRI is an attractive modality for imaging cartilage. Unfortunately, currently available conventional MRI methods are unable to detect the earliest stages of the disease when biochemical changes occur without gross tissue damage (2). Recently, several MRI methods have been proposed to detect PG loss from cartilage (3, 4). In particular, spin-lattice relaxation in the rotating frame (T 1 ) has been demonstrated to be elevated in PG-depleted cartilage (5).T 1 relaxation is sensitive to molecular motions that have correlation times () such that SL ϳ1, where SL ϭ ␥B SL is the strength of the spin-lock field (6). T 1 increases with the strength of the spin-lock field, a phenomenon termed dispersion. T 1 measurements can therefore provide information about the biophysical mechanisms underlying magnetic relaxation. It has been demonstrated that water T 1 relaxation and dispersion (in the 0.1-10 kHz regime) are sensitive to macromolecule-water interactions in protein solutions and possibly also in biological tissues (7-9). Low frequency (0.1-3 kHz) T 1 dispersion has been observed in several systems such as protein solutions (7), bovine articular cartilage (5), human patellar cartilage (10), rodent brain (11), and murine tumor tissue (9). However, the exact nature of T 1 dispersion in biological tissues remains unclear. The range of spin-lock strengths that can be used for in vivo measurements is 0.1-3 kHz (depending on the duration of the spin-lock pulse), without exceeding power deposition limits. Therefore, we have focused our i...