Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins

Head and neck squamous cell carcinoma of unknown primary: Neck dissection and radiotherapy or definitive radiotherapy

Background Management of head and neck carcinoma from unknown primary (HNCUP) remains controversial, with neck dissection and radiotherapy (RT) or definitive RT both commonly used. The purpose of this study was to characterize HNCUP and retrospectively compare outcomes for patients treated with neck dissection + RT versus definitive RT. Methods From 1994 to 2009, 41 patients with HNCUP underwent either neck dissection + RT ( n  = 22) or definitive RT ± concurrent chemotherapy ( n  = 19) at our institution. Treatment outcomes were compared using Kaplan–Meier methods and log‐rank test. Results There were no differences between patients treated with neck dissection + RT and definitive RT in overall survival (OS), progression‐free survival (PFS), locoregional relapse‐free survival (LRFS), freedom from locoregional failure (FFLRG), or freedom from distant failure (FFDF). Among 17 patients who underwent neck dissection + RT for whom human papillomavirus (HPV) status could be determined, HPV(+) patients trended toward improved OS ( p  = .06) and PFS ( p  = .15). Conclusion Neck dissection and postoperative RT resulted in similar outcomes as definitive RT. The prognostic implications of HPV(+) nodes in HNCUP are similar to those in oropharyngeal primary cancers. © 2013 Wiley Periodicals, Inc. Head Neck 36: 1589–1595, 2014Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109313/1/hed23479.pd

Bacterial curli protein promotes the conversion of PAP248-286 into the amyloid SEVI: cross-seeding of dissimilar amyloid sequences

Fragments of prostatic acid phosphatase (PAP248-286) in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection), however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg) acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus) infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide) and Aβ1−40) with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1−40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed

Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

BACKGROUND: We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency. RESULTS: To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion. We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated. CONCLUSION: We propose here an optimized procedure to encapsulate fragile enzymes into liposomes. Optimal encapsulation is achieved by induction of a specific interaction between the enzyme and the lipid surface

A Review of Transcatheter Ablation for the Treatment of AVNRT in Children

Background: Atrioventricular nodal reentrant tachycardia (AVNRT) is an arrhythmia due to re-entrant rhythm within the region of the atrioventricular (AV) node, which accounts for most supraventricular tachycardia (SVT) cases in children. There are two main pathways involved for the re-entrant rhythm, slow and fast pathways, with different anatomic locations and involvement in the circuit associated with AVNRT. AVNRT is rare in newborns, but an increase of prevalence throughout childhood was previously reported.Study Objective/Purpose: Currently, Radiofrequency (RF) ablation is the primary method for the treatment of AVNRT in pediatrics. However, multiple modalities with varying efficacies can also be utilized. In this study, we attempt to review indications and complications of the gold-standard use of RF compared to newer modalities for the ablation treatment of AVNRT in the pediatric population. Results/Discussion: Currently there are two transcatheter ablations methods widely applied as AVNRT treatment: RF ablation and Cryoablation (Cryo). Indications for these methods vary with blood flow in the target area, duration of procedure, and risk of recurrence of AVNRT. Both methods have success rates \u3e90% in AVNRT children, with a 3% complication rate. AV block is the most common complication of RF ablation cases, while Cryo, being a newer technology, requires further investigation. Factors that complicate ablation in AVNRT include anatomical and electrophysiological variations between individuals. Fluoroscopic visualization and 3D-voltage mapping of pathways can provide markers for catheter ablation in AVNRT cases to expedite ablation success and enhance safety. Other predictors of success include: reduced fluoroscopy time, lower patient weight, the ability to induce junctional rhythm in the patient during the procedure, and the utilization of image-based guidance and ice-mapping during the ablation. Conclusion: Radiofrequency catheter ablation remains the highly successful gold standard for the treatment of AVNRT in children, with low complication rates. Cryoablation and other advanced techniques are emerging as new methods tailored to the accessory pathways and more sophisticated structural variations underlying AVNRT in children

β-hairpin-mediated formation of structurally distinct multimers of neurotoxic prion peptides

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A117V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive 'T-state' and a high-affinity active 'R-state' with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ('1-Mut') and five ('5-Mut') mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the KmPyr is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, kcat and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, KmPyr approaches the values it has with FBP and becomes almost temperature independent, kcat increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.</p

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: impact of his-tagging and carotenoid type

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCPs studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and the time scales on which these modifications affect photoactivation. The presence of a His-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP. - Complete functional characterization of Synechocystis and Planktothrix OCPs - Hitherto unknown structures of ECN- and CAN-functionalized Planktothrix OC

Electron diffraction captures high-resolution structures from in vivo protein nanocrystals of Bacillus thuringiensis

Bacillus thuringiensis is one of the most widely used biopesticides worldwide owing to the highly specific pesticidal-proteins various strains produce in the form of nanocrystals. Structure determination of such crystals remains difficult because their small size makes them unsuitable for conventional X-ray crystallography. Here we explore two emerging (cryo-) electron diffraction techniques, namely three-dimensional electron diffraction and serial electron diffraction, as tools for studying the structures of these crystals. Using the mosquitocidal protein Cry11Aa as an example, we compare electron diffraction with state of the art results obtained with an X-ray free electron laser. Our work demonstrates that electron diffraction is a viable alternative for structure determination from such challenging crystals, in some cases outperforming previous results obtained with X-ray free electron lasers. We present a workflow based on readily available instrumentation enabling structure determination directly from the crystals grown in vivo, unperturbed by dissolution and therefore preserved in their native state