Biomedical subjects

Stephane Canaan

Publications and source records attributed to Stephane Canaan.

3 recordsLinked to original sources

PubMed2005

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases.

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.

Amino Acid Sequence↗

PubMed2004

Comparing continuous wave progressive saturation EPR and time domain saturation recovery EPR over the entire motional range of nitroxide spin labels.

The measurement of spin-lattice relaxation rates from spin labels, such as nitroxides, in the presence and absence of spin relaxants provides information that is useful for determining biomolecular properties such as nucleic acid dynamics and the interaction of proteins with membranes. We compare X-band continuous wave (CW) and pulsed or time domain (TD) EPR methods for obtaining spin-lattice relaxation rates of spin labels across the entire range of rotational motion to which relaxation rates are sensitive. Model nitroxides and spin-labeled biological species are used to illustrate the potential complications that arise in extracting relaxation data under conditions typical to biological experiments. The effect of super hyperfine (SHF) structure is investigated for both CW and TD spectra. First and second harmonic absorption and dispersion CW spectra of the nitroxide spin label, TEMPOL, are all fit simultaneously to a model of SHF structure over a range of microwave amplitudes. The CW spectra are novel because all harmonics and microwave phases were acquired simultaneously using our homebuilt CW/TD spectrometer. The effect of the SHF structure on the pulsed free induction decay (FID) and pulsed saturation recovery spectrum is shown for both protonated and deuterated TEMPOL. We present novel pulsed saturation recovery measurements on biological molecules, including spin-lattice relaxation rates of spin-labeled proteins and spin-labeled double-stranded DNA. The impact of structure and dynamics on relaxation rates are discussed in the context of each of these examples. Collisional relaxation rates with oxygen and transition metal paramagnetic relaxants are extracted using both continuous wave and time domain methods. The extent of the errors inherent in the CW method and the advantages of pulsed methods for unambiguously measuring collisional relaxation rates are discussed. Spin-lattice relaxation rates, determined by both CW and pulsed methods, are used to determine the electrostatic potential on the surface of a protein.

DNA↗

PubMed2003

Medium-scale structural genomics: strategies for protein expression and crystallization.

While high-throughput methods of protein production and crystallization are beginning to be well documented, owing to the output of large structural genomics programs, medium-throughput methods at the laboratory scale lag behind. In this paper, we report a possible way for an academic laboratory to adapt high-throughput to medium-throughput methods, on the basis of the first results of two projects aimed at solving the 3D structures of Escherichia coli and Mycobacterium tuberculosis (Tb) proteins of unknown function. We have developed sequential and iterative procedures as well as new technical processes for these programs. Our results clearly demonstrate the value of this medium-throughput approach. For instance, in the first 14 months of the E. coli program, 69 out of 108 target genes led to soluble proteins, 36 were brought to crystallization, and 28 yielded crystals; among the latter, 13 led to usable data sets and 9 to structures. These results, still incomplete, might help in planning future directions of expression and crystallization of proteins applied to medium-throughput structural genomics programs.

Crystallization↗