Biomedical subjects

Ludwig Krippahl

Publications and source records attributed to Ludwig Krippahl.

6 recordsLinked to original sources

PubMed2007

Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen.

The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37 degrees C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the beta-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation intermediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour, which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism.

Amino Acid Motifs↗

PubMed2005

Synechocystis ferredoxin/ferredoxin-NADP(+)-reductase/NADP+ complex: Structural model obtained by NMR-restrained docking.

Ferredoxin (Fd) and ferredoxin-NADP(+)-reductase (FNR) are two terminal physiological partners of the photosynthetic electron transport chain. Based on a nuclear magnetic resonance (NMR)-restrained-docking approach, two alternative structural models of the Fd-FNR complex in the presence of NADP+ are proposed. The protein docking simulations were performed with the software BiGGER. NMR titration revealed a 1:1 stoichiometry for the complex and allowed the mapping of the interacting residues at the surface of Fd. The NMR chemical shifts were encoded into distance constraints and used with theoretically calculated electronic coupling between the redox cofactors to propose experimentally validated docked complexes.

Amino Acid Sequence↗

PubMed2005

Pseudoazurin-nitrite reductase interactions.

The nitrite reductase-binding site on pseudoazurin has been determined by using NMR chemical-shift perturbations. It comprises residues in the hydrophobic patch surrounding the exposed copper ligand His81 as well as several positively charged residues. The binding site is similar for both redox states of pseudoazurin, despite differences in the binding mode. The results suggest that pseudoazurin binds in a well-defined orientation. Docking simulations provide a putative structure of the complex with a binding site on nitrite reductase that has several hydrophobic and polar residues as well as a ridge of negatively charged side chains and a copper-to-copper distance of 14 A.

Azurin↗

PubMed2003

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome.

According to the model proposed in previous papers [Pettigrew, G. W., Prazeres, S., Costa, C., Palma, N., Krippahl, L., and Moura, J. J. (1999) The structure of an electron-transfer complex containing a cytochrome c and a peroxidase, J. Biol. Chem. 274, 11383-11389; Pettigrew, G. W., Goodhew, C. F., Cooper, A., Nutley, M., Jumel, K., and Harding, S. E. (2003) Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans, Biochemistry 42, 2046-2055], cytochrome c peroxidase of Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c(550) at different sites on its molecular surface. Here we use (1)H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c(550) fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c(550) bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c(550) show that the enzyme can accommodate both cytochromes at the same time on adjacent sites. Microcalorimetric titrations are difficult to interpret but are consistent with a weakened binding of horse cytochrome c to a binary complex of cytochrome c peroxidase and cytochrome c(550) and binding of cytochrome c(550) to the cytochrome c peroxidase that is affected little by the presence of horse cytochrome c in the other site. The presence of a substantial capture surface for small cytochromes on the cytochrome c peroxidase has implications for rate enhancement mechanisms which ensure that the two electrons required for re-reduction of the enzyme after reaction with hydrogen peroxide are delivered efficiently.

Animals↗

PubMed2003

A further investigation of the cytochrome b5-cytochrome c complex.

The interaction of reduced rabbit cytochrome b(5) with reduced yeast iso-1 cytochrome c has been studied through the analysis of (1)H-(15)N HSQC spectra, of (15)N longitudinal ( R(1)) and transverse ( R(2)) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b(5) has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b(5).

Animals↗

PubMed2003

Modeling protein complexes with BiGGER.

This article describes the method and results of our participation in the Critical Assessment of PRediction of Interactions (CAPRI) experiment, using the protein docking program BiGGER (Bimolecular complex Generation with Global Evaluation and Ranking) (Palma et al., Proteins 2000;39:372-384). Of five target complexes (CAPRI targets 2, 4, 5, 6, and 7), only one was successfully predicted (target 6), but BiGGER generated reasonable models for targets 4, 5, and 7, which could have been identified if additional biochemical information had been available.

Algorithms↗