Biomedical subjects

S Ealick

Publications and source records attributed to S Ealick.

5 recordsLinked to original sources

PubMed2001

Evidence that NADP+ is the physiological cofactor of ADP-L-glycero-D-mannoheptose 6-epimerase.

ADP-L-glycero-D-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-l-glycero-D-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD(+) cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000) Structure 5, 453-462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD(+), NADP(+), and FAD. In this report we provide data that shows NAD(+) and NADP(+) both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP(+) over NAD(+). The K(d) value for NADP(+) was 26 microm whereas that for NAD(+) was 45 microm. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP(+) had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD(+). Perchloric acid extracts of the purified enzyme were assayed with NAD(+)-specific alcohol dehydrogenase and NADP(+)-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP(+) in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD(+) cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP(+).

Adenine Nucleotides↗

PubMed1997

A system for integrated collection and analysis of crystallographic diffraction data.

A set of linked software modules has been installed at the A1 station of CHESS, the Cornell High Energy Synchrotron Source, which, with the underlying hardware, allows crystallographic users of the facility to evaluate crystals, collect diffraction images and process the images rapidly and with assurance of the quality of the resultant data, thereby making most efficient use of their beam time. The system includes a CCD detector and its controlling software, with a graphical user interface, a convenient oscillation camera featuring automated alignment with the X-ray beam, a program for optimizing crystal rotation range, and the HKL data-reduction package. Principles embodied in this system are applicable to other facilities where crystallographic data are routinely collected.

Journal Article↗

PubMed1996

Crystallization and preliminary X-ray analysis of twinned crystals of a chimeric FK506 binding protein 12 and 13 complexed with FK506.

An FKBPI2/13 chimera with the 80s loop of FKBPI3 replacing the corresponding loop in FKBPI2 tightly binds the immunosuppressive agents FK506 and rapamycin and efficiently catalyzes peptidyl-prolyl cis-trans isomerization. However, the chimera's complex with FK506 does not inhibit calcineurin's phosphatase activity [Yang, Rosen & Schreiber (1993). J. Am. Chem. Soc. 115(2), 819-820]. The chimeric protein crystallizes in space group P1 and the crystals are always twinned. The twin composites are related by a twofold twinning axis parallel to the a axis. A resolution data set (1.5 A resolution) for a twinned crystal was collected at CHESS using 0.91 A X-rays and image plates. Preliminary molecular replacement using data between 15 and 3 A and the FKBPI2-FK506 crystal structure as the search model led to a clear solution with a residual of 34.2%. This 3 A resolution structure provides insight into the structural basis of twinning.

Journal Article↗

PubMed1993

Towards the measurement of ideal data for macromolecular crystallography using synchrotron sources.

Synchrotron radiation has been used extensively to overcome a variety of technical challenges involved in data collection from macromolecular crystals. The next generation of such sources offer a higher brilliance at much shorter wavelengths than hitherto available. Hence, the quality of X-ray diffraction data from crystals of biological macromolecules will be further improved in terms of reduced systematic and random errors, in conjunction with a very high degree of completeness of, and multiple measurements within, the data set. Real data sets should be able to approach closely the quality of ideal data sets. Tests at CHESS are described of the feasibility of recording protein crystal diffraction patterns at ultra-short wavelengths (lambda = 0.3 A) and very-short wavelengths (lambda = 0.5 A), in monochromatic rotating crystal geometry.

Journal Article↗

PubMed1993

A homology model of human interferon alpha-2.

An atomic coordinate five alpha-helix three-dimensional model is presented for human interferon alpha-2 (HuIFN alpha 2). The HuIFN alpha 2 structure was constructed from murine interferon beta (MuIFN beta) by homology modeling using the STEREO and IMPACT programs. The HuIFN alpha 2 model is consistent with its known biochemical and biophysical properties including epitope mapping. Lysine residues predicted to be buried in the model were primarily unreactive with succinimidyl-7-amino-4-methylcoumarin-3-acetic acid (AMCA-NHS), a lysine modification agent, as shown by mass spectrometric analysis of tryptic digests. N-terminal sequence analysis of polypeptides generated by limited digestion of HuIFN alpha 2 with endoproteinase Lys-C demonstrated rapid cleavage at K31, which is consistent with the presence of this residue in a loop in the proposed HuIFN alpha 2 model. Based on this model structure potential receptor binding sites are identified.

Amino Acid Sequence↗