Site Pocket Research Articles

Mutagenic exploration of the active site of lactate dehydrogenase from Plasmodium falciparum

Several site-directed mutations of residues around the active site of the lactate dehydrogenase from Plasmodium falciparum are described. These include changes to three highly, but not completely, conserved residues in the pocket of the active site and also three changes (including deletions) to the active site loop. Changes to residues in the active-site pocket resulted in little or no over-production of protein and no enzymic activity. Likewise, a five residue deletion from the active site loop gave no over-produced protein, while a two residue deletion and changes of residue type in this loop were tolerated. The results are discussed in the light of this protein being a suitable target for novel anti-malarials.

Nonlinear Vibrations in a 2-Dimensional Protein Cluster Model with Linear Bonds

A two dimensional model for the substrate inside a pocket of an active site of an enzyme is presented and investigated as a vibrational system. The parameters of the system are evaluated for α-chymotrypsin. In the case of internal resonance it is analytically and numerically shown that the energy concentrated on a certain degree of freedom might be several times larger than in the non-resonant case. Additionally, the system is driven by harmonic excitations and again energy due to nonlinear phenomena is redistributed inhomogeneously. These results may be of importance for the determination of the rates of catalytic events of substrates bound in pockets of active sites.

The adenine pocket of a single catalytic site is derivatized when the bovine heart mitochondrial F1-ATPase is photoinactivated with 4-amino-1-octylquinaldinium.

The bovine heart mitochondrial F1-ATPase (MF1) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I0.5 value of 48 microM. When irradiated in the presence of AOQ, MF1 is photoinactivated with an apparent Kd of 12 microM. About 1.1 mol of [3H]AOQ were incorporated per mol of MF1 on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF1 photolabeled with [3H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with 3H. Sequence analysis of the labeled peptides revealed that one contained residues 423-441 of the beta subunit. A gap in position 2 of the sequence indicates that beta Phe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342-358 of the beta subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains beta Tyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that beta Tyr345 is labeled in this peptide.

Delayed complications of venous access

Long-term catheter-related complications occur despite the selection of the appropriate device and optimal insertion. Complications typically result from a breakdown in maintenance procedures or from the natural history of long-term venous catheter insertion. In any case, the early detection and management of catheter-related complications may save the device and prolong adequate catheter function. Complications can be divided into mechanical, thrombotic, and infectious categories. Mechanical abnormalities relate to malpositioned tips positioned in undesired locations or against a vessel wall and a loss of the mechanical integrity of the catheter-a fracture. The interventionalist will detect and correct these problems. Thrombotic complications include an intracatheter thrombus, fibrin sheath, and catheter-related venous thrombosis. Each requires management which many involve intracatheter urokinase, balloon sheath disruption or intravenous lysis, respectively. Infectious complications manifest as simple exit site infections, a tunnel or pocket abscess, or catheter-related sepsis. Antibiotics with or without incision and drainage, catheter exchange, or catheter removal may be needed. The inserting service (interventional radiologist) must take responsibility for the detection and management of these catheter-related complications.

Threonine 81 of the trp repressor of Escherichia coli plays an auxiliary role in the formation of the corepressor binding pocket.

A mutational study was performed on the corepressor (L-tryptophan) binding site of the trp repressor of Escherichia coli. Threonine 81, one of the residues forming the hydrophobic pocket of the binding site, was replaced with Ser, Cys and Met by cassette mutagenesis. Biochemical characterization showed that all these mutations caused a moderate decrease in tryptophan binding activity (free energy change approximately 1 kcal/mol). The results suggested that the binding pocket is rather flexible in the vicinity of Thr81. On the other hand, the mutations produced a discernible decrease in the repressor activity in vivo, apparently by weakening or eliminating the hydrogen bond between Thr81 and the operator DNA, as well as by introducing possible side-chain rearrangement.

Glycine 85 of the trp-repressor of E. coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches.

Experimental and computational analyses were performed on the corepressor (L-tryptophan) binding site of the trp-repressor of Escherichia coli to investigate the ligand-protein interactions. Gly85, one of the residues forming the hydrophobic pocket of the binding site, was systematically replaced with Ala, Val, Leu and Trp by cassette mutagenesis. Biochemical characterization showed that all these mutations caused significant decreases in tryptophan binding activity. Free energy perturbation calculations were performed for the mutants and were consistent with the experimental results. The lack of a side chain at position 85 was concluded to be essential for binding the corepressor; the structure of the binding pocket was suggested to be tight in the vicinity of Gly85.

Prediction of peptide affinity to HLA DRB1*0401.

Hydrogen bonding between conserved amino acids in the HLA DR and the peptide backbone of the ligand both provide the majority of free energy of binding and force the peptide ligands to adopt a similar extended conformation. Consequently the corresponding side chains of all peptides interact with similar pockets in the binding site. For peptides of a common length the contribution of the peptide backbone can be treated as a constant and the differential affinity can be viewed as a simple sum of the side chain interactions. These can be quantified by measuring the effects of each of the naturally occurring amino acids in the context of a simplified polyalanine backbone containing an aromatic amino acid to orient the peptide unequivocally in the binding site. The dataset of the relative contributions can be used to predict quantitatively the affinity of any peptide sequence.