Anders Bergkvist

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties

Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.

NMR CHARACTERIZATION OF THE NADP(H)-BINDING DOMAIN OF ESCHERICHIA COLI TRANSHYDROGENASE : SEQUENTIAL ASSIGNMENT AND GLOBAL FOLD

The soluble NADP(H)‐binding domain of Escherichia coli transhydrogenase (186 amino acids, 20.4 kDa, rotational correlation time 14 ns) was characterized using NMR techniques. The global fold is similar to that of a classical dinucleotide‐binding fold with six parallel β‐strands in a central sheet surrounded by helices and irregular structures, but is lacking both αD and αE. The substrate is bound in an extended conformation at the C‐terminal end of the parallel β‐sheet and our data support the notion of a redox dependent structural rearrangement.

Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by 1H-NMR chemical shift analysis

A combination of site‐directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side‐chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the 1H chemical shifts by use of two‐dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild‐type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild‐type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.

Fast algorithms for finding disjoint subsequences with extremal densities

We derive fast algorithms for the problem of finding, on the real line, a prescribed number of intervals of maximum total length that contain at most some prescribed number of points from a given point set. Basically this is a typical dynamic programming problem, however, for input sizes much bigger than the two parameters we can improve the obvious time bound by selecting a restricted set of candidate intervals that are sufficient to build some optimal solution. As a byproduct, the same idea improves an algorithm for a similar subsequence problem recently brought up by Chen, Lu and Tang at IWBRA 2005. The problems are motivated by the search for significant patterns in certain biological data. While the algorithmic idea for the asymptotic worst-case bound is rather evident, we also consider further heuristics to save even more time in typical instances. One of them, described in this paper, leads to an apparently open problem of computational geometry flavour (where we are seeking a subquadratic algorithm) which might be interesting in itself.