Search results for "Mutant"

A stochastic model of mutant growth

1987

Targeting Cavity-Creating p53 Cancer Mutations with Small-Molecule Stabilizers: the Y220X Paradigm

2020

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pock…

Identification of residues in the putative 5th helical region of human interleukin-6, important for activation of the IL-6 signal transducer, gp130

1996

AbstractWe have previously shown that L58 in the putative 5th helical region of human interleukin-6 (IL-6) is important for activation of the IL-6 signal transducer gp130 [de Hon et al. (1995) FEBS Lett. 369, 187–191]. To further explore the importance of individual residues in this region for gp130 activation we have now combined Ala substitutions of residues E52, S53, S54, K55, E56, L58 and E60 with other substitutions in IL-6, known to affect gp130 activation (Q160E and T163P). The combination mutant protein with L58A completely lost the capacity to induce the proliferation of XG-1 myeloma cells, and could effectively antagonize wild type IL-6 activity on these cells. Moreover, the data …

Conformational control of Bax localization and apoptotic activity by Pro168.

2004

In healthy cells, Bax resides inactive in the cytosol because its COOH-terminal transmembrane region (TMB) is tucked into a hydrophobic pocket. During apoptosis, Bax undergoes a conformational change involving NH2-terminal exposure and translocates to mitochondria to release apoptogenic factors. How this process is regulated remains unknown. We show that the TMB of Bax is both necessary and sufficient for mitochondrial targeting. However, its availability for targeting depends on Pro168 located within the preceding loop region. Pro168 mutants of Bax lack apoptotic activity, cannot rescue the apoptosis-resistant phenotype of Bax/Bak double knockout cells, and are retained in the cytosol even…

Molecular dynamics studies on HIV-1 protease: a comparison of the flap motions between wild type protease and the M46I/G51D double mutant

2007

The emergence of drug-resistant mutants of HIV-1 is a tragic effect associated with conventional long-treatment therapies against acquired immunodeficiency syndrome. These mutations frequently involve the aspartic protease encoded by the virus; knowledge of the molecular mechanisms underlying the conformational changes of HIV-1 protease mutants may be useful in developing more effective and longer lasting treatment regimes. The flap regions of the protease are the target of a particular type of mutations occurring far from the active site. These mutations modify the affinity for both substrate and ligands, thus conferring resistance. In this work, molecular dynamics simulations were perform…

Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome.

2020

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nanoto milliseconds. We identify two sequentially forming Lumi-R states …

Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase

2008

Molecular simulations based on the use of hybrid quantum mechanics/molecular mechanics methods are able to provide detailed information about the complex enzymatic reactions and the consequences of specific mutations on the activity of the enzyme. In this work, the reduction of pyruvate to lactate catalysed by wild-type and Asp168Ala mutant lactate dehydrogenase (LDH) has been studied by means of simulations using a very flexible molecular model consisting of the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules. Our results indicate that the Asp168Ala mutation provokes a shift in the p K a value of Glu199 that becomes unprotonated at n…

Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains II and III in specificity towards Spodoptera exigua larvae

2004

Several mutants of the Bacillus thuringiensis Cry1Ca toxin affected with regard to specific activity towards Spodoptera exigua were studied. Alanine was used to replace single residues in loops 2 and 3 of domain II (mutant pPB19) and to replace residues 541– 544 in domain III (mutant pPB20). Additionally, a Cry1Ca mutant combining all mutations was constructed (mutant pPB21). Toxicity assays showed a marked decrease in toxicity against S. exigua for all mutants, while they retained their activity against Manduca sexta, confirming the importance of these residues in determining insect specificity. Parameters for binding to the specific receptors in BBMV (brush border membrane vesicles) of S.…

Pore formation by Vibrio cholerae cytolysin follows the same archetypical mode as beta-barrel toxins from gram-positive organisms.

2009

Vibrio cholerae cytolysin (VCC) forms SDS-stable heptameric beta-barrel transmembrane pores in mammalian cell membranes. In contrast to structurally related pore formers of gram-positive organisms, no oligomeric prepore stage of assembly has been detected to date. In the present study, disulfide bonds were engineered to tie the pore-forming amino acid sequence to adjacent domains. In their nonreduced form, mutants were able to bind to rabbit erythrocytes and to native erythrocyte membranes suspended in PBS solution and form SDS-labile oligomers. These remained nonfunctional and represented the long-sought VCC prepores. Disulfide bond reduction in these oligomers released the pore-forming se…

Crystal structures of bR(D85S) favor a model of bacteriorhodopsin as a hydroxyl-ion pump

2003

AbstractStructural features on the extracellular side of the D85S mutant of bacteriorhodopsin (bR) suggest that wild-type bR could be a hydroxyl-ion pump. A position between the protonated Schiff base and residue 85 serves as an anion-binding site in the mutant protein, and hydroxyl ions should have access to this site during the O-intermediate of the wild-type bR photocycle. The guanidinium group of R82 is proposed (1) to serve as a shuttle that eliminates the Born energy penalty for entry of an anion into this binding pocket, and conversely, (2) to block the exit of a proton or a related proton carrier.