Binding of bis-ANS to Bacillus subtilis lipase: A combined computational and experimental investigation

Kamal, Md. Zahid; Ali, Jamshaid; Rao, Nalam Madhusudhana

doi:10.1016/j.bbapap.2013.04.021

Cited by 7 publications

(3 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The same assay principle ( Figure 2A ) applies to 1-anilinonaphtalene-8-sulphonate (ANS) accumulation because the fluorescence quantum yield increases when ANS binds to hydrophobic structures (e.g., proteins, membranes) in the cell. ANS is a substrate of MexD and has been used to study this transporter in P. aeruginosa ( Walmsley et al, 1994 ; Mao et al, 2002 ; Kamal et al, 2013 ). Another example is 1,2′-dinaphthylamine which has been used to measure AcrB eﬄux in E. coli because it is a substrate of AcrB and becomes strongly fluorescent when it partitions into a phospholipid bilayer ( Bohnert et al, 2011a ).…”

Section: Eﬄux Assaysmentioning

confidence: 99%

Interaction of antibacterial compounds with RND eﬄux pumps in Pseudomonas aeruginosa

Dreier

Ruggerone²

2015

Front. Microbiol.

143

126

View full text Add to dashboard Cite

Pseudomonas aeruginosa infections are becoming increasingly difficult to treat due to intrinsic antibiotic resistance and the propensity of this pathogen to accumulate diverse resistance mechanisms. Hyperexpression of eﬄux pumps of the Resistance-Nodulation-Cell Division (RND)-type multidrug eﬄux pumps (e.g., MexAB-OprM), chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA) is often detected in clinical isolates and contributes to worrying multi-drug resistance phenotypes. Not all antibiotics are affected to the same extent by the aforementioned RND eﬄux pumps. The impact of eﬄux on antibiotic activity varies not only between different classes of antibiotics but also between members of the same family of antibiotics. Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by eﬄux activity. The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries. This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

show abstract

Section: Eﬄux Assaysmentioning

confidence: 99%

Interaction of antibacterial compounds with RND eﬄux pumps in Pseudomonas aeruginosa

Dreier

Ruggerone²

2015

Front. Microbiol.

143

126

View full text Add to dashboard Cite

show abstract

“…10,25 Bis-ANS binding sites found by docking, validated with steady-state and time-resolved fluorescence assays, have been used to identify hydrophobic patches in a lipase from Bacillus subtilis . 26 …”

Section: Introductionmentioning

confidence: 99%

Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin

Khago¹,

Wong²,

Kingsley³

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

View full text Add to dashboard Cite

Background The objective of this study was to determine whether the cataract-related G18V variant of human γS-crystallin has increased exposure of hydrophobic residues that could explain its aggregation propensity and/or recognition by αB-crystallin. Methods We used an ANS fluorescence assay and NMR chemical shift perturbation to experimentally probe exposed hydrophobic surfaces. These results were compared to flexible docking simulations of ANS molecules to the proteins, starting with the solution-state NMR structures of γS-WT and γS-G18V. Results γS-G18V exhibits increased ANS fluorescence, suggesting increased exposed hydrophobic surface area. The specific residues involved in ANS binding were mapped by NMR chemical shift perturbation assays, revealing ANS binding sites in γS-G18V that are not present in γS-WT. Molecular docking predicts three binding sites that are specific to γS-G18V corresponding to the exposure of a hydrophobic cavity located at the interdomain interface, as well as two hydrophobic patches near a disordered loop containing solvent-exposed cysteines, all but one of which is buried in γS-WT. Conclusions Although both proteins display non-specific binding, more residues are involved in ANS binding to γS-G18V, and the affected residues are localized in the N-terminal domain and the nearby interdomain interface, proximal to the mutation site. General Significance Characterization of changes in exposed hydrophobic surface area between wild-type and variant proteins can help elucidate the mechanisms of aggregation propensity and chaperone recognition, presented here in the context of cataract formation. Experimental data and simulations provide complementary views of the interactions between proteins and the small molecule probes commonly used to study aggregation.

show abstract

“…3b ). To further probe global structural changes in CitAP-BsLA we employed the fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) 38 39 , which binds to hydrophobic surface patches of proteins 40 . Upon dye binding, an increased fluorescence emission as well as a blue-shift of the emission maximum, compared to the free dye, can be observed.…”

Section: Resultsmentioning

confidence: 99%

A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase

Kaschner

Schillinger

Fettweiss

et al. 2017

Sci Rep

View full text Add to dashboard Cite

Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme’s active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae. The site of domain fusion was rationally determined based on whole-protein site-saturation mutagenesis data, complemented by computational evolutionary-coupling analyses. Functional assays, combined with biochemical and biophysical studies suggest a mechanism for control, similar but distinct to the one of the parent CitA protein, with citrate acting as an indirect modulator of Triton-X100 inhibition of the fusion protein. Our study demonstrates that the introduction of ligand-dependent regulatory control by domain fusion is surprisingly facile, suggesting that the catalytic mechanism of some enzymes may be evolutionary optimized in a way that it can easily be perturbed by small conformational changes.

show abstract

Binding of bis-ANS to Bacillus subtilis lipase: A combined computational and experimental investigation

Cited by 7 publications

References 39 publications

Interaction of antibacterial compounds with RND eﬄux pumps in Pseudomonas aeruginosa

Interaction of antibacterial compounds with RND eﬄux pumps in Pseudomonas aeruginosa

Increased hydrophobic surface exposure in the cataract-related G18V variant of human γS-crystallin

A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase

Contact Info

Product

Resources

About