Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTD and CreCat) to be structurally independent. Amide 15N relaxation measurements of the CreCat domain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein–protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site. Moreover, binding to loxP DNA induces a conformational change that dislodges the C terminus, resulting in a cis-to-trans switch that is likely to enable protein–protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency.
Mechanistic understanding of DNA recombination in the Cre-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution NMR to discover the link between intrinsic flexibility and function in Cre recombinase. TROSY-NMR spectra show the N-terminal and C-terminal catalytic domains (Cre NTD , Cre Cat ) to be structurally independent. Amide 15 N relaxation measurements of the Cre Cat domain reveal fast time scale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the Cterminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity via trans protein-protein interactions, is unexpectedly rigid in free Cre.Chemical shift perturbations and intra-and inter-molecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative auto-inhibitory conformation for the αN region of free Cre, wherein it packs in cis over the protein DNA binding surface and active site.Moreover, binding to loxP DNA induces a conformational change that dislodge the C-terminus, resulting in a cis to trans switch that is likely to enable protein-protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a re-examination of the mechanisms by which this widely-utilized gene editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency. SIGNIFICANCE STATEMENTThe Cre-loxP system is a widely used gene editing tool that has enabled transformative advances in immunology, neuroscience and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage.Remarkably, we found that in the absence of DNA the C-terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein-protein interactions required for assembly of recombinogenic Cre intasomes.
macromolecules in serum. Analytical ultracentrifugation (AUC) is one of the experimental methods best suited to determine and understand these factors. Sedimentation velocity, sedimentation equilibrium, synthetic boundary, and band sedimentation experiments are performed in order to determine the behavior of therapeutic antibodies in highly concentrated environments. The Aviv-FDS utilizes a single fluorescently labeled sedimenting species against a concentrated and heterogeneous background of serum proteins which results in nonideality. Nonideality results from excluded volume and charge effects. This describes the hydrodynamic and thermodynamic nonideality of a single component as a function of high concentrations of other components in solution. Thus, the aim of our current work is to study the behavior of a fluorescently labeled therapeutic antibody in the presence of high concentrations of human serum proteins (HSA and human IgG) and in human serum. To study and understand the behavior of therapeutic antibodies in the presence of HSA, human IgG, and other serum components, tracer experiments are done pairwise as a function of HSA, IgG and therapeutic protein concentration. This generates a 3x3 matrix of data that describes self-and cross-term hydrodynamic and thermodynamic nonideality (K s and BM1). The goal is to develop a preclinical biophysical method for quantitative hydrodynamic and thermodynamic analysis of therapeutic proteins in crowded environments like serum. Cre recombinase (38.5 kDa, 343 residues), belonging to the tyrosine recombinase family of enzymes, mediates site-specific recombination between specific loxP DNA recognition sequences. Two Cre molecules bind to a 34 bp inverted loxP sequence and further assemble with another Cre 2 -loxP complex to form an antiparallel tetrameric synaptic complex. Crystal structures of the synaptic tetrad show that the DNA duplexes bend by about 80 facilitating the protein-protein interactions between Cre protomers. While single molecule and ensemble kinetics data implicate protein conformational changes (i.e., dynamics) in the progression of the Cre-loxP recombination pathway, the motions, timescales, amplitudes, and their structural basis still remain poorly understood. We aim to characterize the dynamics of Cre recombinase at atomic resolution using solution NMR spectroscopy through measurements of backbone and side chain heteronuclear relaxation. Backbone 15 N T 1 , T 2 and { 1 H}-15 N heteronuclear NOE relaxation measurements on the free catalytic domain of Cre identified specific regions that exhibit dynamics on the ps-ns timescales. Two of the flexible regions, the b2-b3 loop and helix aN, form part of the inter-protomer interface in the synaptic tetramer, while a third, the aJ-aK loop, contacts the DNA phosphate backbone. These findings shed light on the solution structure of unbound Cre recombinase and reinforce the hypothesis that protein dynamics are significant for mediating DNA recognition and synaptic assembly. Progress towards characterizing dynam...
Background: Antimicrobial agents (AMAs) are the mainstay of treatment in modern medicine. In view of emerging threat of antimicrobial resistance, there is a requirement aimed at identifying patterns of antimicrobial prescribing. This study focuses on the prescribing pattern of AMAs and the incidence of adverse drug reaction (ADR) among the inpatients of Malabar Medical College and Hospital, Calicut. Aims and Objectives: The objectives of this study were to evaluate antibiotics prescribing pattern and ADR’s in a tertiary health-care center in Northern Kerala. Materials and Methods: This is a prospective and observational study for a period was 6 months between March 2023 and August 2023. A total of 500 enrolled patients were observed from admission till discharge. Medical case sheets, drug charts, and laboratory investigations were recorded in a self-designed standardized performa and analyzed. Results: Most of the patients were in the age group 51 to 60 (26%), followed by 61 to 70 (22%), followed by 71 to 80 (16%). Out of 500 patients assessed, 64% were males and 36% were females. Out of 100 prescriptions, 64% of the prescriptions were in the parenteral form, 30% oral, and the remaining in topical form. Out of 500 prescriptions, 83% used brand name while prescribing, while the remaining 13 % used generic name. The most commonly prescribed antibiotic was Cefoperazone-Sulbactem combination (16%) followed by Piparacillin, Tazobactam, and Azithromycin Combination. Out of 500 patients, 30 patients (6%) developed ADR. The most common ADRs seen with the study included hypersensitivity (44%), gastrointestinal upset (30%), and headaches (10%). Conclusion: From an analysis of 500 patients from six in-patient departments, it was found that hospital doctors prescribed antibiotics more logically, with fewer newer drugs and no banned ones. ADRs were seen in 6% of patients, which was comparatively higher compared to previous studies.
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