Rapid and simple protein-stability screens: application to membrane proteins

Yeh, Andrew; McMillan, Andrew W.; Stowell, Michael H. B.

doi:10.1107/s0907444906005233

Cited by 64 publications

(66 citation statements)

References 37 publications

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“…The fluorescence data were plotted, normalized, and the first derivative of the curve calculated to provide the melting temperature (T m ) using GraphPad Prism 5.0 as detailed in Ref. 28. The change in melting temperature (⌬T m ) was calculated as the difference between the T m of the mutant construct in the presence of fatty acid compared with the mutant construct alone.…”

Section: Methodsmentioning

confidence: 99%

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Koszelak-Rosenblum

Krol

Simmons

et al. 2008

Journal of Biological Chemistry

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Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid ␣-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in K m , whereas no substantial change in K m was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/ R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14 -20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.Pathogen attack on plants brings about the activation of multiple enzyme systems that results in the production of oxylipins from 18 -22 carbon fatty acid precursors. The generation of these bioactive lipid mediators initiates and sustains the defense reaction of the plant against insects, bacteria, fungi, and other pathogens (1, 2). One of the enzymes up-regulated during the host defense response is pathogen-inducible oxygenase (PIOX), 2 which catalyzes a non-lipoxygenase type of fatty acid oxygenation (3). PIOX belongs to a larger family of heme-containing proteins that oxygenate fatty acids (4), which include the mammalian cyclooxygenases (COX-1 and COX-2; (5)), linoleate diol synthase (LDS) from the fungus Gaeumannomyces graminis (6, 7), and a Pseudomonas alcalignes protein of unknown function encoded by OrfX (8). PIOX has also been identified in many plant species, including Nicotiana attenuata (9), Nicotiana tabacum (3), Arabidopsis thaliana (3, 10), O. sativa (11), Capsicum annuum (12), and Lycopersicon esculentum (13).PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous deca...

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Section: Methodsmentioning

confidence: 99%

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Koszelak-Rosenblum

Krol

Simmons

et al. 2008

Journal of Biological Chemistry

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“…DSF uses a hydrophobic dye which increases in fluorescence intensity upon binding to hydrophobic pockets in proteins that become accessible during heating (57). Thermal denaturation curves recorded by DSF for each of the serotypes as VLPs and rAAV-GFP samples showed clear differences in the transition temperature.…”

Section: Stability Of Aav Capsidsmentioning

confidence: 99%

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Rayaprolu

Kruse

Kant

et al. 2013

J Virol

128

108

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bIcosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.

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“…The thermal stability of GCase (Cerezyme) in the absence and presence of increasing concentrations of IFG was determined using differential scanning fluorimetry (DSF) by monitoring the increase in emission intensity of NanoOrange, [18][19][20]29] an environmentally sensitive fluorescent probe that undergoes fluorescence enhancement upon exposure to the hydrophobic interior of a protein. As the temperature is elevated, the fluorescence intensity of NanoOrange increases as a larger proportion of the GCase in solution unfolds, enabling binding of the probe to the hydrophobic interior.…”

Section: Ifg Enhances Global Stability and Activitymentioning

confidence: 99%

Isofagomine Induced Stabilization of Glucocerebrosidase

Kornhaber¹,

Tropak

Maegawa

et al. 2008

ChemBioChem

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Structurally destabilizing mutations in acid β-glucosidase (GCase) can result in Gaucher disease (GD). The iminosugar isofagomine (IFG), a competitive inhibitor and a potential pharmacological chaperone of GCase, is currently undergoing clinical evaluation for the treatment of GD. An X-ray crystallographic study of the GCase-IFG complex revealed a hydrogen bonding network between IFG and certain active site residues. It was suggested that this network may translate into greater global stability. Here it is demonstrated that IFG does increase the global stability of wild-type GCase, shifting its melting curve by ~15 °C and that it enhances mutant GCase activity in pretreated N370S/N370S and F213I/L444P patient fibroblasts. Additionally, amide hydrogen/ deuterium exchange mass spectroscopy (H/D-Ex) was employed to identify regions within GCase that undergo stabilization upon IFG-binding. H/D-Ex data indicate that the binding of IFG not only restricts the local protein dynamics of the active site, but also propagates this effect into surrounding regions.

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Rapid and simple protein-stability screens: application to membrane proteins

Cited by 64 publications

References 37 publications

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Isofagomine Induced Stabilization of Glucocerebrosidase

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