Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution. AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations. Three optical systems are available for the analytical ultracentrifuge (absorbance, interference and fluorescence) that permit precise and selective observation of sedimentation in real time. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules in complex mixtures and at high solute concentrations. In sedimentation velocity, the movement of solutes in high centrifugal fields is interpreted using hydrodynamic theory to define the size, shape and interactions of macromolecules. Sedimentation equilibrium is a thermodynamic method where equilibrium concentration gradients at lower centrifugal fields are analyzed to define molecule mass, assembly stoichiometry, association constants and solution nonideality. Using specialized sample cells and modern analysis software, researchers can use sedimentation velocity to determine the homogeneity of a sample and define whether it undergoes concentration-dependent association reactions. Subsequently, more thorough model-dependent analysis of velocity and equilibrium experiments can provide a detailed picture of the nature of the species present in solution and their interactions.

The T cell immunoglobulin mucin (Tim) family of receptors regulates effector CD4(+) T cell functions and is implicated in autoimmune and allergic diseases. Tim-3 induces immunological tolerance, and engagement of the Tim-3 immunoglobulin variable (IgV) domain by galectin-9 is important for appropriate termination of T helper 1-immune responses. The 2 A crystal structure of the Tim-3 IgV domain demonstrated that four cysteines, which are invariant within the Tim family, form two noncanonical disulfide bonds, resulting in a surface not present in other immunoglobulin superfamily members. Biochemical and biophysical studies demonstrated that this unique structural feature mediates a previously unidentified galectin-9-independent binding process and suggested that this structural feature is conserved within the entire Tim family. The current work provided a graphic example of the relationship between sequence, structure, and function and suggested that the interplay between multiple Tim-3-binding activities contributes to the regulated assembly of signaling complexes required for effective Th1-mediated immunity.

Spastin, an AAA ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. Many other AAA proteins form ring-shaped hexamers and contain pore loops, which project into the ring's central cavity and act as ratchets that pull on target proteins, leading, in some cases, to conformational changes. We show that Spastin assembles into a hexamer and that loops within the central pore recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. We also show that Spastin contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Given that Spastin engages the MT in two places and that both interactions are required for severing, we propose that severing occurs by forces exerted on the C-terminal tail of tubulin, which results in a conformational change in tubulin, which releases it from the polymer.

SummaryPKR (protein kinase R) is a central component of the interferon antiviral defense pathway. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase. PKR then phosphorylates eIF2α, thus inhibiting protein synthesis in virally-infected cells. Here, we define the mechanism of PKR activation using a series dsRNAs of increasing length. A minimal dsRNA of 30 bp is required to bind two PKR monomers and 30 bp is the smallest dsRNA that elicits autophosphorylation activity. Thus, the ability of dsRNAs to function as PKR activators is correlated with binding of two or more PKR monomers. Sedimentation velocity data fit a model where PKR monomers sequentially attach to a single dsRNA. These results support an activation mechanism where the role of the dsRNA is to bring two or more PKR monomers in close proximity to enhance dimerization via the kinase domain. This model explains the inhibition observed at high dsRNA concentrations and the strong dependence of maximum activation on dsRNA binding affinity. Binding affinities increase dramatically upon reducing the salt concentration from 200 to 75 mM NaCl and we observe that a second PKR can bind to the 20 bp dsRNA. Nonspecific assembly of PKR on dsRNA occurs stochastically without apparent cooperativity.

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