Hauptman Woodward Institute
Hauptman Woodward Institute
Sundlov J.A.,Hauptman Woodward Institute |
Sundlov J.A.,State University of New York at Buffalo |
Fontaine D.M.,Connecticut College |
Southworth T.L.,Connecticut College |
And 3 more authors.
Biochemistry | Year: 2012
Beetle luciferases catalyze a two-step reaction that includes the initial adenylation of the luciferin substrate, followed by an oxidative decarboxylation that ultimately produces light. Evidence for homologous acyl-CoA synthetases supports a domain alternation catalytic mechanism in which these enzymes C-terminal domain rotates by ∼140° to adopt two conformations that are used to catalyze the two partial reactions. While many structures exist of acyl-CoA synthetases in both conformations, to date only biochemical evidence supports domain alternation with luciferase. We have determined the structure of a cross-linked luciferase enzyme that is trapped in the second conformation. This new structure supports the role of the second catalytic conformation and provides insights into the biochemical mechanism of the luciferase oxidative step. © 2012 American Chemical Society.
PubMed | Connecticut College and Hauptman Woodward Institute
Type: | Journal: Photochemistry and photobiology | Year: 2016
Unlike the enchanting yellow-green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H-bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination.
Pryor Jr. E.E.,Membrane Protein Structural Biology Consortium |
Pryor Jr. E.E.,University of Virginia |
Horanyi P.S.,Membrane Protein Structural Biology Consortium |
Horanyi P.S.,University of Virginia |
And 17 more authors.
Science | Year: 2013
Posttranslational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CAAX sequence motifs (where A is an aliphatic residue and X is any residue). Isoprenylation is followed by cleavage of the AAX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CAAX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active site and substrate-binding groove. The cavity is accessible to the external milieu by means of gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds by means of a processive mechanism of substrate insertion, translocation, and ejection.
Huang R.,Hauptman Woodward Institute |
Huang R.,Cornell University
Journal of Synchrotron Radiation | Year: 2011
One of the challenges of tuning bimorph mirrors with many electrodes is that the calculated focusing voltages can be different by more than the safety limit (such as 500 V for the mirrors used at 17-ID at the Advanced Photon Source) between adjacent electrodes. A study of this problem at 17-ID revealed that the inverse problem of the tuning in situ, using X-rays, became ill-conditioned when the number of electrodes was large and the calculated focusing voltages were contaminated with measurement errors. Increasing the number of beamlets during the tuning could reduce the matrix condition number in the problem, but obtaining voltages with variation below the safety limit was still not always guaranteed and multiple iterations of tuning were often required. Applying Tikhonov regularization and using the L-curve criterion for the determination of the regularization parameter made it straightforward to obtain focusing voltages with well behaved variations. Some characteristics of the tuning results obtained using Tikhonov regularization are given in this paper. © 2011 International Union of Crystallography Printed in Singapore - all rights reserved.
Chaudhuri B.N.,Hauptman Woodward Institute |
Chaudhuri B.N.,State University of New York at Buffalo |
Dean R.,Hauptman Woodward Institute
Journal of Molecular Biology | Year: 2011
The bacterial chromosome trafficking apparatus or the segrosome participates in the mitotic-like segregation of the chromosomes prior to cell division in several bacteria. ParB, which is the parS DNA-binding component of the segrosome, polymerizes on the parS-adjacent chromosome to form a nucleoprotein filament of unknown nature for the segregation function. We combined static light scattering, circular dichroism and small-angle X-ray scattering to present evidence that the apo form of the mycobacterial ParB forms an elongated dimer with intrinsically disordered regions as well as folded domains in solution. A comparison of the solution scattering of the apo and the parS-bound ParBs indicates a rather drastic compaction of the protein upon DNA binding. We propose that this binding-induced conformational transition is priming the ParB for polymerization on the DNA template. © 2011 Elsevier Ltd. All rights reserved.
Demonte D.,State University of New York at Buffalo |
Drake E.J.,Hauptman Woodward Institute |
Lim K.H.,State University of New York at Buffalo |
Lim K.H.,Johns Hopkins University |
And 3 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2013
We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20-40% improvement in the measured koff. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t1/2) from 11 to 83 min at 20°C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. © 2013 Wiley Periodicals, Inc.
Mitchell C.A.,Hauptman Woodward Institute |
Mitchell C.A.,State University of New York at Buffalo |
Tucker A.C.,University of Georgia |
Escalante-Semerena J.C.,University of Georgia |
And 2 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2015
The adenosine monoposphate-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ∼110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain. © 2014 Wiley Periodicals, Inc.
Huang R.,Hauptman Woodward Institute |
Meron M.,University of Chicago |
Kujala N.,Illinois Institute of Technology |
Barrea R.A.,Illinois Institute of Technology
Journal of Synchrotron Radiation | Year: 2010
Micro-focusing optical devices at synchrotron beamlines usually have a limited acceptance, but more flux can be intercepted if such optics are used to focus secondary sources created by the primary optics. Flux throughput can be maximized by placing the secondary focusing optics close to or exactly at the secondary source position. However, standard methods of beamline optics analysis, such as the lens equation or matching the mirror surface to an ellipse, work poorly when the source-to-optics distance is very short. In this paper the general characteristics of the focusing of beams with Gaussian profiles by a thin lens are analysed under the paraxial approximation in phase space, concluding that the focusing of a beam with a short source-to-optics distance is distinct from imaging the source; slope errors are successfully included in all the formulas so that they can be used to calculate beamline focusing with good accuracy. A method is also introduced to use the thin-lens result to analyse the micro-focusing produced by an elliptically bent trapezoid-shaped Kirkpatrick-Baez mirror. The results of this analysis are in good agreement with ray-tracing simulations and are confirmed by the experimental results of the secondary focusing at the 18-ID Bio-CAT beamline (at the APS). The result of secondary focusing carried out at 18-ID using a single-bounce capillary can also be explained using this phase-space analysis. A discussion of the secondary focusing results is presented at the end of this paper. © 2010 International Union of Crystallography.
Huang R.,Hauptman Woodward Institute |
Meron M.,University of Chicago
Nuclear Instruments and Methods in Physics Research, Section A: Accelerators, Spectrometers, Detectors and Associated Equipment | Year: 2013
The techniques for the cooling of the first crystal of a monochromator are by now mature and are used routinely to deal with the heat loads resulting from the intense beams generated by third generation synchrotron insertion device sources. However, the thermal stability of said monochromators, which crucially depends on proper shielding of X-ray scattering off the first crystal, remains a serious consideration. This will become even more so in the near future, as many synchrotron facilities are upgrading to higher beam currents and energies. During a recent upgrade of the 17-ID beamline at the APS it was recognized that accurate simulation of the spatial distribution of the power scattered off the first crystal was essential for the understanding and remediation of the observed large temperature increase of the first crystal's scattering shield. The calculation is complex, due to the broad energy spectrum of the undulator and the prevalence of multiple X-ray scattering events within the bulk of the crystal, thus the Monte Carlo method is the natural tool for such a task. A successful simulation was developed, for the purpose of the 17-ID upgrade, and used to significantly improve the design of the first crystal's scattering shield. © 2013 Elsevier B.V.
PubMed | Hauptman Woodward Institute
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2016
The Gram-negative pathogen Pseudomonas aeruginosa uses a nonribosomal peptide synthetase (NRPS) biosynthetic cluster for the production of a peptide siderophore. In addition to four multimodular NRPS proteins, the biosynthetic pathway also requires several additional enzymes involved in the production of nonproteinogenic amino acids and maturation of the peptide product. Among the proteins that are required for the final steps in pyoverdine synthesis is PvdN, a pyridoxal phosphate-dependent enzyme that catalyzes an uncharacterized step in pyoverdine production. This study reports the high-resolution structure of PvdN bound to a PLP cofactor solved by multi-wavelength anomalous dispersion (MAD). The PvdN model shows high structural homology to type I aspartate aminotransferases and also contains positive density that suggests an uncharacterized external aldimine.