Determination of protein backbone structures from residual dipolar couplings.

There are a number of circumstances in which a focus on determination of the backbone structure of a protein, as opposed to a complete all-atom structure, may be appropriate. This is particularly the case for structures determined as a part of a structural genomics initiative in which computational modeling of many sequentially related structures from the backbone of a single family representative is anticipated. It is, however, also the case when the backbone may be a stepping-stone to more targeted studies of ligand interaction or protein-protein interaction. Here an NMR protocol is described that can produce a backbone structure of a protein without the need for extensive experiments directed at side chain resonance assignment or the collection of structural information on side chains. The procedure relies primarily on orientational constraints from residual dipolar couplings as opposed to distance constraints from NOEs. Procedures for sample preparation, data acquisition, and data analysis are described, along with examples from application to small target proteins of a structural genomics project.

Modeling temporal behavior of postnatal cat retinal ganglion cells.

During development, mammalian retinal ganglion cells (RGCs) go through marked ontogenetic changes with respect to their excitable membrane properties. Voltage-clamp studies conducted in our laboratory have shown that the amplitude, voltage-dependence and kinetics of activation and inactivation (where present) of Na(+), K(+) and Ca(2+) conductances all exhibit developmental changes during a time when the firing patterns of mammalian ganglion cells shift from being transient to being predominantly sustained in nature. In order to better understand the contribution of each conductance to the generation of spikes and spiking patterns, we have developed a model based on our experimental data. For simplicity, we have initially used experimental data obtained from postnatal ganglion cells. At this age the ontogenetic changes observed in the characteristics of the various ionic currents are complete. Utilizing the methods adopted by Hodgkin and Huxley for the giant squid axon, we have determined rate equations for the activation and inactivation properties of the I(A), I(K dr), I(Na), I(Ca L), I(Ca N), and I(leak) currents in postnatal cat RGCs. Combining these with a simplified model of the calcium-activated potassium current (I(KCa)), we have solved and analysed the resulting differential equations. While spikes and spiking patterns resembling experimental data could be obtained from a model in which [Ca(2+)i] was averaged across the whole cell, more accurate simulations were obtained when the diffusion of intracellular Ca(2+) was modeled spatially. The resulting spatial calcium gradients were more effective in gating I(KCa), and our simulations more accurately matched the recorded amplitude and shape of individual spikes as well as the frequency of maintained discharges observed in mammalian postnatal RGCs.