X-ray Crystallographic Characterization of the Co(II)-substituted Tris-bound Form of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e

The X-ray crystal structure of the Co(II)-loaded form of the aminopeptidase from Aeromonas proteolytica ([CoCo(AAP)]) was solved to 2.2 Å resolution. [CoCo(AAP)] folds into an α/β globular domain with a twisted β-sheet hydrophobic core sandwiched between α-helices, identical to [ZnZn(AAP)]. Co(II) binding to AAP does not introduce any major conformational changes to the overall protein structure and the amino acid residues ligated to the dicobalt(II) cluster in [CoCo(AAP)] are the same as those in the native Zn(II)-loaded structure with only minor perturbations in bond lengths. The Co(II)–Co(II) distance is 3.3 Å. Tris(hydroxymethyl)aminomethane (Tris) coordinates to the dinuclear Co(II) active site of AAP with one of the Tris hydroxyl oxygen atoms (O4) forming a single oxygen atom bridge between the two Co(II) ions. This is the only Tris atom coordinated to the metals with Co1–O and Co2–O bonds distances of 2.2 and 1.9 Å, respectively. Each of the Co(II) ions resides in a distorted trigonal bipyramidal geometry. This important structure bridges the gap between previous structural and spectroscopic studies performed on AAP and is discussed in this context

A Parallel Manipulator with Only Translational Degrees of Freedom

This report presents a novel three degree of freedom parallel manipulator that employs only revolute joints and constrains the manipulator output to translational motion. Closed-form solutions are developed for both the inverse and forward kinematics. It is shown that the inverse kinematics problem has up to four real solutions, and the forward kinematics problem has up to 16 real solutions

Inhibition of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e by l-Leucinephosphonic Acid. Spectroscopic and Crystallographic Characterization of the Transition State of Peptide Hydrolysis

The nature of the interaction of the transition-state analogue inhibitor l-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a Ki of 6.6 μM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 Å resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an η-1,2-μ-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed

Unique Sex-Based Approach Identifies Transcriptomic Biomarkers Associated with Non-Syndromic Craniosynostosis

Background The premature fusion of one cranial suture, also referred to as non-syndromic craniosynostosis, most commonly involves premature fusion of the sagittal, coronal, or metopic sutures, in that order. Population-based epidemiological studies have found that the birth prevalence of single-suture craniosynostosis is both suture- and sex-dependent. Methods Transcriptomic data from 199 individuals with isolated sagittal (n = 100), unilateral coronal (n = 50), and metopic (n = 49) synostosis were compared against a control population (n = 50) to identify transcripts accounting for the different sex-based frequencies observed in this disease. Results Differential sex-based gene expression was classified as either gained (divergent) or lost (convergent) in affected individuals to identify transcripts related to disease predilection. Divergent expression was dependent on synostosis sub-type, and was extensive in metopic craniosynostosis specifically. Convergent microarray-based expression was independent of synostosis sub-type, with convergent expression of FBN2, IGF2BP3, PDE1C and TINAGL1 being the most robust across all synostosis sub-types. Conclusions Analysis of sex-based gene expression followed by validation by qRT-PCR identified that concurrent upregulation of FBN2 and IGF2BP3 , and downregulation of TINAGL1 in craniosynostosis cases were all associated with increased RUNX2 expression and may represent a transcriptomic signature that can be used to characterize a subset of single-suture craniosynostosis cases

Spectroscopic and X-ray Crystallographic Characterization of Bestatin Bound to the Aminopeptidase from \u3cem\u3eAeromonas (Vibrio) proteolytica\u3c/em\u3e

Binding of the competitive, slow-binding inhibitor bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from Aeromonas proteolytica (AAP) was examined by both spectroscopic and crystallographic methods. Electronic absorption spectra of the catalytically competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the presence of bestatin revealed that both of the divalent metal ions in AAP are involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum of the [CoCo(AAP)]−bestatin complex exhibited no observable perpendicular- or parallel-mode signal. These data indicate that the two CoII ions in AAP are antiferromagnetically coupled yielding an S = 0 ground state and suggest that a single oxygen atom bridges between the two divalent metal ions. The EPR data obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]−bestatin complex was solved to 2.0 Å resolution. Both side chains of bestatin occupy a well-defined hydrophobic pocket that is adjacent to the dinuclear ZnII active site. The amino acid residues ligated to the dizinc(II) cluster in AAP are identical to those in the native structure with only minor perturbations in bond length. The alkoxide oxygen of bestatin bridges between the two ZnII ions in the active site, displacing the bridging water molecule observed in the native [ZnZn(AAP)] structure. The M−M distances observed in the AAP−bestatin complex and native AAP are identical (3.5 Å) with alkoxide oxygen atom distances of 2.1 and 1.9 Å from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 Å. In addition, the NH2 group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 Å. A combination of the spectroscopic and X-ray crystallographic data presented herein with the previously reported mechanistic data for AAP has provided additional insight into the substrate-binding step of peptide hydrolysis as well as insight into important small molecule features for inhibitor design

Optimization of a Three DOF Translational Platform for Well- Conditioned Workspace

Two optimization studies on the design of a three degree of freedom translational parallel platform are conducted and the results are compared. The objective function of the first study maximizes total volume of the manipulator workspace without regard to the quality of the workspace. The second study optimizes the total volume of well conditioned workspace by maximizing a global condition index. The global condition index is a function of the condition number of the Jacobian matrix, providing a means of measuring the amplification error between the actuators and the end effector. Both objective functions involve an integration over the workspace of the manipulator. This integral is approximated using the Monte Carlo method

Differential Expression of Extracellular Matrix-Mediated Pathways in Single-Suture Craniosynostosis

Craniosynostosis is a disease defined by premature fusion of one or more cranial sutures. The mechanistic pathology of single-suture craniosynostosis is complex and while a number of genetic biomarkers and environmental predispositions have been identified, in many cases the causes remain controversial and inconclusive. In this study, gene expression data from 199 patients with isolated sagittal (n = 100), unilateral coronal (n = 50), and metopic (n = 49) synostosis are compared against both a control population (n = 50), as well as each other. After controlling for variables contributing to potential bias, FGF7, SFRP4, and VCAM1 emerged as genes associated with single-suture craniosynostosis due to their significantly large changes in gene expression compared to the control population. Pathway analysis implicated focal adhesion and extracellular matrix (ECM)-receptor interaction as differentially regulated gene networks when comparing all cases of single-suture synostosis and controls. Lastly, overall gene expression was found to be highly conserved between coronal and metopic cases, as evidenced by the fact that WNT2 and IGFBP2 were the only genes differentially regulated to a significantly large extent in a direct comparison. The identification of genes and gene networks associated with Fgf/Igf/Wnt signaling and ECM-mediated focal adhesion not only support the involvement of biomarkers previously reported to be related to craniosynostosis, but also introduce novel transcripts and pathways that may play critical roles in its pathogenesis

Ferromagnetic phase transition and Bose-Einstein condensation in spinor Bose gases

Phase transitions in spinor Bose gases with ferromagnetic (FM) couplings are studied via mean-field theory. We show that an infinitesimal value of the coupling can induce a FM phase transition at a finite temperature always above the critical temperature of Bose-Einstein condensation. This contrasts sharply with the case of Fermi gases, in which the Stoner coupling $I_s$ can not lead to a FM phase transition unless it is larger than a threshold value $I_0$. The FM coupling also increases the critical temperatures of both the ferromagnetic transition and the Bose-Einstein condensation.Comment: 4 pages, 4 figure