scholarly journals Engineering a light-controlled F1ATPase using structure-based protein design

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2286 ◽  
Author(s):  
Daniel Hoersch
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Atp Hydrolysis ◽  
Site Specific ◽  
Dynamic Constraint ◽  
End Distance ◽  
A Site ◽  
Hydrolysis Activity

The F1sub-complex of ATP synthase is a biological nanomotor that converts the free energy of ATP hydrolysis into mechanical work with an astonishing efficiency of up to 100% (Kinosita et al., 2000). To probe the principal mechanics of the machine, I re-engineered the active site ofE.coliF1ATPase with a structure-based protein design approach: by incorporation of a site-specific, photoswitchable crosslinker, whose end-to-end distance can be modulated by illumination with light of two different wavelengths, a dynamic constraint was imposed on the inter-atomic distances of the α and β subunits. Crosslinking reduced the ATP hydrolysis activity of four designs tested in vitro and in one case created a synthetic ATPase whose activity can be reversibly modulated by subsequent illumination with near UV and blue light. The work is a first step into the direction of the long-term goal to design nanoscaled machines based on biological parts that can be precisely controlled by light.

scholarly journals Pentapeptide repeat proteins QnrB1 and AlbG require ATP hydrolysis to rejuvenate poisoned gyrase complexes

2020 ◽  
Author(s):  
Łukasz Mazurek ◽  
Dmitry Ghilarov ◽  
Elizabeth Michalczyk ◽  
Zuzanna Pakosz ◽  
Wojciech Czyszczoń ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Type Ii ◽  
Site Specific ◽  
Repeat Proteins ◽  
Gyrase A ◽  
Microcin B17 ◽  
Long Term Studies

ABSTRACTDNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of “poisons”, namely natural product toxins (e.g. albicidin. microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We compared activities of two such proteins, QnrB1 and AlbG in vitro. Each of them provided specific protection against its cognate toxin (fluoroquinolone or albicidin), which strictly required ATP hydrolysis by gyrase. Through a combination of fluorescence anisotropy, pull-downs and photocrosslinking we show that QnrB1 binds to the GyrB protein. We further probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped strong and specific crosslinks to the N-terminal ATPase/transducer domain. We propose a model in which protective PRPs bind to the enzyme as T-segment DNA mimics to promote dissociation of the bound poison molecule.

scholarly journals Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

2002 ◽  
Vol 70 (2) ◽  
pp. 787-793 ◽  
Author(s):  
Patricia Guerry ◽  
Christine M. Szymanski ◽  
Martina M. Prendergast ◽  
Thomas E. Hickey ◽  
Cheryl P. Ewing ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Phase Variation ◽  
Outer Core ◽  
Gene Encoding ◽  
Site Specific ◽  
Reading Frame ◽  
Specific Mutation ◽  
Normal Human ◽  
A Site

ABSTRACT The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM2 and GM3 gangliosides, with minor amounts of structures mimicking GD1b and GD2. Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM2 to GM3 ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM3 ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

Long-term evaluation of coal fly ash and mine tailings co-placement: A site-specific study

2009 ◽  
Vol 91 (1) ◽  
pp. 237-244 ◽  
Author(s):  
Muluken B. Yeheyis ◽  
Julie Q. Shang ◽  
Ernest K. Yanful
Keyword(s):  
Fly Ash ◽  
Mine Tailings ◽  
Coal Fly Ash ◽  
Site Specific ◽  
Specific Study ◽  
Term Evaluation ◽  
A Site

scholarly journals The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

2019 ◽  
Author(s):  
Cody M. Rogers ◽  
Chun-Ying Lee ◽  
Samuel Parkins ◽  
Nicholas J. Buehler ◽  
Sabine Wenzel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage Response ◽  
Nuclease Activity ◽  
Site Specific ◽  
Damage Response ◽  
Physical Barriers ◽  
A Site ◽  
Translesion Polymerases ◽  
Stimulation Of

AbstractDNA inter-strand crosslink (ICL) repair requires a complicated network of DNA damage response pathways. Removal of these lesions is vital as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principle mechanism for ICL repair in metazoans and is coupled to replication. In Saccharomyces cerevisiae, a degenerate FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease that is hypothesized to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked RECQL4, as a novel component of Pso2- mediated ICL repair. Here, we show that Hrq1 stimulates the Pso2 nuclease in a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulates Pso2 translesion nuclease activity through a site- specific ICL in vitro. Stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these dangerous lesions.

scholarly journals Measurement and modelling of evaporation from a coastal wetland in Maputaland, South Africa

2012 ◽  
Vol 9 (2) ◽  
pp. 1741-1782 ◽  
Author(s):  
A. D. Clulow ◽  
C. S. Everson ◽  
M. G. Mengistu ◽  
C. Jarmain ◽  
G. P. W. Jewitt ◽  
...  
Keyword(s):  
Water Balance ◽  
Animal Species ◽  
Coastal Wetland ◽  
Surface Renewal ◽  
Site Specific ◽  
Combination Of Methods ◽  
A Site ◽  
Sr Method ◽  
Subtropical Environment

Abstract. The contribution of freshwater supply from the Mfabeni Mire to Lake St. Lucia during dry periods is important to the survival of certain plant and animal species in the iSimangaliso Wetland Park. This freshwater supply is mainly dependent on the variability of the major components of the water balance, namely rainfall and total evaporation (ET). Attempts to quantify the water balance have been limited through uncertainties in quantifying ET from the Mfabeni Mire. Despite advances in evaporation measurement and modelling from wetlands, there still exists some doubt as to which methods are best suited to characterise wetland ET with most authors suggesting a combination of methods. In this study, the surface renewal (SR) method was successfully used to determine the long-term ET (12 months) from the Mfabeni Mire with calibration using eddy covariance during two window periods of approximately one week each. The SR method was found to be inexpensive, reliable and with low power requirements for unattended operation. The annual ET was lower (900 mm yr−1) than expected, due to cloud cover in summer and low atmospheric demand throughout the year, despite the available water and high windspeeds. Daily ET estimates were compared to the Priestley-Taylor results and a site specific calibration α = 1.0 was obtained for the site. The Priestley-Taylor results agreed well with the actual ET from the surface renewal technique (R2 = 0.96) throughout the 12 month period. A monthly crop factor (Kc) was determined for the standardised FAO-56 Penman-Monteith. However, Kc was variable in some months and should be used with caution for daily ET modelling. These results represent not only some of the first long-term measurements of ET from a wetland in Southern Africa, but also one of the few studies of actual ET in a subtropical peatland in the Southern Hemisphere. The study provides wetland ecologists and hydrologists with guidelines for the use of two internationally applied models for the estimation of wetland ET within a coastal, subtropical environment.

scholarly journals Transport of Alzheimer’s associated amyloid-β catalyzed by P-glycoprotein

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250371
Author(s):  
James W. McCormick ◽  
Lauren Ammerman ◽  
Gang Chen ◽  
Pia D. Vogel ◽  
John G. Wise
Keyword(s):  
Cell Culture ◽  
Atp Hydrolysis ◽  
Amyloid Β ◽  
Md Simulations ◽  
Membrane Transporter ◽  
Biochemical Activity ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Hydrolysis Activity

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer’s disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer’s associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

scholarly journals Thermo-Sensitive Vesicles in Controlled Drug Delivery for Chemotherapy

Pharmaceutics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 150 ◽  
Author(s):  
Elisabetta Mazzotta ◽  
Lorena Tavano ◽  
Rita Muzzalupo
Keyword(s):  
Drug Delivery ◽  
Cancer Treatment ◽  
Controlled Drug Delivery ◽  
Solid Tumours ◽  
Site Specific ◽  
Mild Hyperthermia ◽  
Promising Tool ◽  
A Site

Thermo-sensitive vesicles are a promising tool for triggering the release of drugs to solid tumours when used in combination with mild hyperthermia. Responsivity to temperature makes them intelligent nanodevices able to provide a site-specific chemotherapy. Following a brief introduction concerning hyperthermia and its advantageous combination with vesicular systems, recent investigations on thermo-sensitive vesicles useful for controlled drug delivery in cancer treatment are reported in this review. In particular, the influence of bilayer composition on the in vitro and in vivo behaviour of thermo-sensitive formulations currently under investigation have been extensively explored.

scholarly journals Attempts to develop an enzyme converting DHIV to KIV

2019 ◽  
Vol 32 (6) ◽  
pp. 261-270
Author(s):  
Kenji Oki ◽  
Frederick S Lee ◽  
Stephen L Mayo
Keyword(s):  
Protein Design ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Mandelate Racemase ◽  
Acid Dehydratase ◽  
Enolase Superfamily ◽  
Beta Elimination ◽  
Cost Efficient ◽  
Hydrolysis Activity

Abstract Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.

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