Physico-chemical and biological analysis of true combinatorial libraries

Lambert, Pierre‐Herve; Bertin, Sophie; Volland, Jean‐Paul; Fauchère, Jean‐Luc

doi:10.1016/s0378-4347(98)00569-6

Cited by 12 publications

(12 citation statements)

References 143 publications

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“…Five b-strands define one side of the molecule whereas a small a2helix (58-65) spans three of these strands: (3)(4)(5)(6)(7)(8), (72-77), (99-109) on the other side. Residues belonging to the other three b-strands (22)(23)(24)(25)(26)(27)(28)(29)(30)(31), (35)(36)(37)(38)(39), (46)(47)(48)(49)(50), the helix and two long loops (51-57), (80-98) form a hydrophobic binding pocket that harbors rapamycin. Hydrogen bonds that rapamycin establishes with residues D37, Q53, I56, Y82 are conserved both in the recombinant and synthetic calstabin structures [ Fig.…”

Section: Structural Studymentioning

confidence: 99%

“…In our company, we entered the peptide chemistry domain a long time ago through various approaches (see e.g., [21][22][23] ), and only recently did we decide to explore the possibility of producing, refolding, characterizing, and even crystallizing synthetic proteins. The goal behind this program was and still is to master those technologies in our laboratory, in order to explore several hard-to-produce enzymes, to modify Biologics to render them more stable, as well as to launch some structure/mutations relationship studies on target enzymes implicated in key physiopathological pathways.…”

Section: Introductionmentioning

confidence: 99%

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Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6)

Bacchi

Jullian²,

Sirigu

et al. 2016

Protein Science

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Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structurefunction relationship of enzymes reachable by complete chemical synthesis.

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Section: Structural Studymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6)

Bacchi

Jullian²,

Sirigu

et al. 2016

Protein Science

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“…The majority of quantitative studies of AHLs, however, have focused on planktonic cultures using a variety of AHL±reporter constructs such as an Agrobacterium tumefaciens strain (Shaw et al, 1997). Other non-AHL components in the extract may also interfere with the reporter system (Boutin et al, 1999). However, quantitative bioassays that are currently in use are limited in both analyte separation and detection.…”

Section: Introductionmentioning

confidence: 99%

“…For example, sensitivity for the A. tumefaciens assay ranges from 0.0005 pmol for the endogenous signal N-3-oxo-octanoyl homoserine lactone (OOHL) to 300 pmol for hexanoyl homoserine lactone (Shaw et al, 1997). Other non-AHL components in the extract may also interfere with the reporter system (Boutin et al, 1999). In the only quantitative assay reported in the literature that is not based on a bioassay, Reimmann et al (1997) reported the use of high-performance liquid chromatography (HPLC)-UV, although no sensitivity or other validation data were provided.…”

Section: Introductionmentioning

confidence: 99%

A novel and sensitive method for the quantification of N‐3‐oxoacyl homoserine lactones using gas chromatography–mass spectrometry: application to a model bacterial biofilm

Charlton¹,

Nys²,

Netting³

et al. 2000

Environmental Microbiology

297

231

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A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).

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“…cal i ntera cti on i n condensed m atter [2,3]. In tho se cases when a form ed com pound canno t b e i sol ated, PCA i s the onl y metho d capabl e o f pro duci ng com p ound ' s comp ositi on.…”

mentioning

confidence: 99%

Application of the Electro-Optical Kerr Effect in Physical-Chemical Analysis of Binary Systems

Prezhdo

Τyurin²,

Pisarczyk

et al. 2002

Acta Phys. Pol. A

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Physico-chemical and biological analysis of true combinatorial libraries

Cited by 12 publications

References 143 publications

Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6)

Total chemical synthesis, refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6)

A novel and sensitive method for the quantification of N‐3‐oxoacyl homoserine lactones using gas chromatography–mass spectrometry: application to a model bacterial biofilm

Application of the Electro-Optical Kerr Effect in Physical-Chemical Analysis of Binary Systems

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