Weik M

General

Full name : Weik Martin

First name : Martin

Mail : Institut de Biologie Structurale\; 41 rue Jules Horowitz\; Grenoble\; 38027

Zip Code :

City :

Country : France

Email : weik@ibs.fr

Phone : +33 4 38 78 95 80

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References (38)

Title : Rivastigmine and metabolite analogues with putative Alzheimer's disease-modifying properties in a Caenorhabditis elegans model. - Dighe_2019_Commun.Chem_2_35
Author(s) : Dighe SN , De la Mora E , Chan S , Kantham S , McColl G , Miles JA , Veliyath SK , Sreenivas BY , Nassar ZD , Silman I , Sussman JL , Weik M , McGeary RP , Parat MO , Brazzolotto X , Ross BP
Ref : Communications chemistry , 2 :35 , 2019
Abstract : The development of polyphenols as drugs for Alzheimer's disease (AD) is thwarted by their meagre brain availability due to instability and poor druglikeness. Here we describe the successful development of stable, druglike polyphenolic analogues of the current AD drug rivastigmine, that have high apparent blood-brain barrier permeabilities and multifunctional properties for AD treatment. The compounds inhibit cholinesterases and amyloid beta (Abeta) fibrillation, protect against Abeta42-induced toxicity in vitro, and demonstrate efficacy in vivo in a transgenic Caenorhabditis elegans model expressing Abeta42, with potencies similar to rivastigmine and natural polyphenols. The results suggest that a tertiary amine substituent is amenable for developing water-soluble, membrane-permeable polyphenols, and its incorporation adjacent to a hydroxy group is favourable for intramolecular hydrogen bonding that facilitates membrane permeability. Carbamylation of one hydroxy group protects the polyphenols from degradation and mostly improves their membrane permeability. These design strategies may assist in the development of polyphenol-based drugs.
ESTHER : Dighe_2019_Commun.Chem_2_35
PubMedSearch : Dighe_2019_Commun.Chem_2_35
PubMedID:
Gene_locus related to this paper: human-BCHE , torca-ACHE

Title : Novel multitarget-directed ligands targeting acetylcholinesterase and sigma1 receptors as lead compounds for treatment of Alzheimer's disease: Synthesis, evaluation, and structural characterization of their complexes with acetylcholinesterase - Lalut_2019_Eur.J.Med.Chem_162_234
Author(s) : Lalut J , Santoni G , Karila D , Lecoutey C , Davis A , Nachon F , Silman I , Sussman JL , Weik M , Maurice T , Dallemagne P , Rochais C
Ref : Eur Journal of Medicinal Chemistry , 162 :234 , 2019
Abstract : Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously inhibit acetylcholinesterase and act as an agonist of the 5-HT4 receptor, which displays promising activities in vivo. Pharmacomodulation of donecopride allowed us to develop a novel series of indole derivatives possessing interesting in vitro activities toward AChE and the sigma1 receptor. The crystal structures of complexes of the most promising compounds with Torpedo californica AChE were solved in order to further understand their mode of inhibition.
ESTHER : Lalut_2019_Eur.J.Med.Chem_162_234
PubMedSearch : Lalut_2019_Eur.J.Med.Chem_162_234
PubMedID: 30447434
Gene_locus related to this paper: torca-ACHE

Title : Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents - Santoni_2018_J.Med.Chem_61_7630
Author(s) : Santoni G , de Sousa J , De la Mora E , Dias J , Jean L , Sussman JL , Silman I , Renard PY , Brown RCD , Weik M , Baati R , Nachon F
Ref : Journal of Medicinal Chemistry , 61 :7630 , 2018
Abstract : Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.
ESTHER : Santoni_2018_J.Med.Chem_61_7630
PubMedSearch : Santoni_2018_J.Med.Chem_61_7630
PubMedID: 30125110
Gene_locus related to this paper: torca-ACHE

Title : Potent 3-Hydroxy-2-Pyridine Aldoxime Reactivators of Organophosphate-Inhibited Cholinesterases with Predicted Blood-Brain Barrier Penetration - Zorbaz_2018_Chemistry_24_9675
Author(s) : Zorbaz T , Braiki A , Marakovic N , Renou J , De la Mora E , Macek Hrvat N , Katalinic M , Silman I , Sussman JL , Mercey G , Gomez C , Mougeot R , Perez B , Baati R , Nachon F , Weik M , Jean L , Kovarik Z , Renard PY
Ref : Chemistry , 24 :9675 , 2018
Abstract : A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.
ESTHER : Zorbaz_2018_Chemistry_24_9675
PubMedSearch : Zorbaz_2018_Chemistry_24_9675
PubMedID: 29672968
Gene_locus related to this paper: torca-ACHE

Title : Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP - Colletier_2016_J.Phys.Chem.Lett_7_882
Author(s) : Colletier JP , Sliwa M , Gallat FX , Sugahara M , Guillon V , Schiro G , Coquelle N , Woodhouse J , Roux L , Gotthard G , Royant A , Uriarte LM , Ruckebusch C , Joti Y , Byrdin M , Mizohata E , Nango E , Tanaka T , Tono K , Yabashi M , Adam V , Cammarata M , Schlichting I , Bourgeois D , Weik M
Ref : J Phys Chem Lett , 7 :882 , 2016
Abstract : Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.
ESTHER : Colletier_2016_J.Phys.Chem.Lett_7_882
PubMedSearch : Colletier_2016_J.Phys.Chem.Lett_7_882
PubMedID: 26866390

Title : Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability - Fraser_2016_J.Mol.Biol_428_2359
Author(s) : Fraser NJ , Liu JW , Mabbitt PD , Correy GJ , Coppin CW , Lethier M , Perugini MA , Murphy JM , Oakeshott JG , Weik M , Jackson CJ
Ref : Journal of Molecular Biology , 428 :2359 , 2016
Abstract : Oligomerization has been suggested to be an important mechanism for increasing or maintaining the thermostability of proteins. Although it is evident that protein-protein contacts can result in substantial stabilization in many extant proteins, evidence for evolutionary selection for oligomerization is largely indirect and little is understood of the early steps in the evolution of oligomers. A laboratory-directed evolution experiment that selected for increased thermostability in the alphaE7 carboxylesterase from the Australian sheep blowfly, Lucilia cuprina, resulted in a thermostable variant, LcalphaE7-4a, that displayed increased levels of dimeric and tetrameric quaternary structure. A trade-off between activity and thermostability was made during the evolution of thermostability, with the higher-order oligomeric species displaying the greatest thermostability and lowest catalytic activity. Analysis of monomeric and dimeric LcalphaE7-4a crystal structures revealed that only one of the oligomerization-inducing mutations was located at a potential protein-protein interface. This work demonstrates that by imposing a selective pressure demanding greater thermostability, mutations can lead to increased oligomerization and stabilization, providing support for the hypothesis that oligomerization is a viable evolutionary strategy for protein stabilization.
ESTHER : Fraser_2016_J.Mol.Biol_428_2359
PubMedSearch : Fraser_2016_J.Mol.Biol_428_2359
PubMedID: 27016206
Gene_locus related to this paper: luccu-E3aest7

Title : The role of protein dynamics in the evolution of new enzyme function - Campbell_2016_Nat.Chem.Biol_12_944
Author(s) : Campbell EC , Kaltenbach M , Correy GJ , Carr PD , Porebski BT , Livingstone EK , Afriat-Jurnou L , Buckle AM , Weik M , Hollfelder F , Tokuriki N , Jackson CJ
Ref : Nat Chemical Biology , 12 :944 , 2016
Abstract : Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
ESTHER : Campbell_2016_Nat.Chem.Biol_12_944
PubMedSearch : Campbell_2016_Nat.Chem.Biol_12_944
PubMedID: 27618189

Title : Mapping the Accessible Conformational Landscape of an Insect Carboxylesterase Using Conformational Ensemble Analysis and Kinetic Crystallography - Correy_2016_Structure_24_977
Author(s) : Correy GJ , Carr PD , Meirelles T , Mabbitt PD , Fraser NJ , Weik M , Jackson CJ
Ref : Structure , 24 :977 , 2016
Abstract : The proper function of enzymes often depends upon their efficient interconversion between particular conformational sub-states on a free-energy landscape. Experimentally characterizing these sub-states is challenging, which has limited our understanding of the role of protein dynamics in many enzymes. Here, we have used a combination of kinetic crystallography and detailed analysis of crystallographic protein ensembles to map the accessible conformational landscape of an insect carboxylesterase (LcalphaE7) as it traverses all steps in its catalytic cycle. LcalphaE7 is of special interest because of its evolving role in organophosphate insecticide resistance. Our results reveal that a dynamically coupled network of residues extends from the substrate-binding site to a surface loop. Interestingly, the coupling of this network that is apparent in the apoenzyme appears to be reduced in the phosphorylated enzyme intermediate. Altogether, the results of this work highlight the importance of protein dynamics to enzyme function and the evolution of new activity.
ESTHER : Correy_2016_Structure_24_977
PubMedSearch : Correy_2016_Structure_24_977
PubMedID: 27210287
Gene_locus related to this paper: luccu-E3aest7

Title : Correlation of the dynamics of native human acetylcholinesterase and its inhibited huperzine A counterpart from sub-picoseconds to nanoseconds - Trapp_2014_J.R.Soc.Interface_11_
Author(s) : Trapp M , Tehei M , Trovaslet M , Nachon F , Martinez N , Koza MM , Weik M , Masson P , Peters J
Ref : J R Soc Interface , 11 : , 2014
Abstract : It is a long debated question whether catalytic activities of enzymes, which lie on the millisecond timescale, are possibly already reflected in variations in atomic thermal fluctuations on the pico- to nanosecond timescale. To shed light on this puzzle, the enzyme human acetylcholinesterase in its wild-type form and complexed with the inhibitor huperzine A were investigated by various neutron scattering techniques and molecular dynamics simulations. Previous results on elastic neutron scattering at various timescales and simulations suggest that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. In the work presented here, the focus was laid on quasi-elastic (QENS) and inelastic neutron scattering (INS). These techniques give access to different kinds of individual diffusive motions and to the density of states of collective motions at the sub-picoseconds timescale. Hence, they permit going beyond the first approach of looking at mean square displacements. For both samples, the autocorrelation function was well described by a stretched-exponential function indicating a linkage between the timescales of fast and slow functional relaxation dynamics. The findings of the QENS and INS investigation are discussed in relation to the results of our earlier elastic incoherent neutron scattering and molecular dynamics simulations.
ESTHER : Trapp_2014_J.R.Soc.Interface_11_
PubMedSearch : Trapp_2014_J.R.Soc.Interface_11_
PubMedID: 24872501

Title : Reaction site-driven regioselective synthesis of AChE inhibitors - Oueis_2014_Org.Biomol.Chem_12_156
Author(s) : Oueis E , Santoni G , Ronco C , Syzgantseva O , Tognetti V , Joubert L , Romieu A , Weik M , Jean L , Sabot C , Nachon F , Renard PY
Ref : Org Biomol Chem , 12 :156 , 2014
Abstract : The enzyme-directed synthesis is an emerging fragment-based lead discovery approach in which the biological target is able to assemble its own multidentate ligands from a pool of building blocks. Here, we report for the first time the use of the human acetylcholinesterase (AChE) as an enzyme for the design and synthesis of new potent heterodimeric huprine-based inhibitors. Both the specific click chemistry site within the protein and the regioselectivity of the Huisgen cycloaddition observed suggest promising alternatives in the design of efficient mono- and dimeric ligands of AChE. Finally, a detailed computational modelling of the click reaction was conducted to further understand the origin of this TGS selectivity.
ESTHER : Oueis_2014_Org.Biomol.Chem_12_156
PubMedSearch : Oueis_2014_Org.Biomol.Chem_12_156
PubMedID: 24216754

Title : Design, synthesis and biological evaluation of novel tetrahydroacridine pyridine- aldoxime and -amidoxime hybrids as efficient uncharged reactivators of nerve agent-inhibited human acetylcholinesterase - Kliachyna_2014_Eur.J.Med.Chem_78C_455
Author(s) : Kliachyna M , Santoni G , Nussbaum V , Renou J , Sanson B , Colletier JP , Arboleas M , Loiodice M , Weik M , Jean L , Renard PY , Nachon F , Baati R
Ref : Eur Journal of Medicinal Chemistry , 78C :455 , 2014
Abstract : A series of new uncharged functional acetylcholinesterase (AChE) reactivators including heterodimers of tetrahydroacridine with 3-hydroxy-2-pyridine aldoximes and amidoximes has been synthesized. These novel molecules display in vitro reactivation potencies towards VX-, tabun- and paraoxon-inhibited human AChE that are superior to those of the mono- and bis-pyridinium aldoximes currently used against nerve agent and pesticide poisoning. Furthermore, these uncharged compounds exhibit a broader reactivity spectrum compared to currently approved remediation drugs.
ESTHER : Kliachyna_2014_Eur.J.Med.Chem_78C_455
PubMedSearch : Kliachyna_2014_Eur.J.Med.Chem_78C_455
PubMedID: 24704618

Title : Relation between dynamics, activity and thermal stability within the cholinesterase family - Trovaslet_2013_Chem.Biol.Interact_203_14
Author(s) : Trovaslet M , Trapp M , Weik M , Nachon F , Masson P , Tehei M , Peters J
Ref : Chemico-Biological Interactions , 203 :14 , 2013
Abstract : Incoherent neutron scattering is one of the most powerful tools for studying dynamics in biological matter. Using the cold neutron backscattering spectrometer IN16 at the Institut Laue Langevin (ILL, Grenoble, France), temperature dependence of cholinesterases' dynamics (human butyrylcholinesterase from plasma: hBChE; recombinant human acetylcholinesterase: hAChE and recombinant mouse acetylcholinesterase: mAChE) was examined using elastic incoherent neutron scattering (EINS). The dynamics was characterized by the averaged atomic mean square displacement (MSD), associated with the sample flexibility at a given temperature. We found MSD values of hAChE above the dynamical transition temperature (around 200K) larger than for mAChE and hBChE, implying that hAChE is more flexible than the other ChEs. Activation energies for thermodynamical transition were extracted through the frequency window model (FWM) (Becker et al. 2004) [1] and turned out to increase from hBChE to mAChE and finally to hAChE, inversely to the MSDs relations. Between 280 and 316K, catalytic studies of these enzymes were carried out using thiocholine esters: at the same temperature, the hAChE activity was systematically higher than the mAChE or hBChE ones. Our results thus suggest a strong correlation between dynamics and activity within the ChE family. We also studied and compared the ChEs thermal inactivation kinetics. Here, no direct correlation with the dynamics was observed, thus suggesting that relations between enzyme dynamics and catalytic stability are more complex. Finally, the possible relation between flexibility and protein ability to grow in crystals is discussed.
ESTHER : Trovaslet_2013_Chem.Biol.Interact_203_14
PubMedSearch : Trovaslet_2013_Chem.Biol.Interact_203_14
PubMedID: 22940283

Title : Structure and function of an insect alpha-carboxylesterase (alphaEsterase7) associated with insecticide resistance - Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
Author(s) : Jackson CJ , Liu JW , Carr PD , Younus F , Coppin C , Meirelles T , Lethier M , Pandey G , Ollis DL , Russell RJ , Weik M , Oakeshott JG
Ref : Proc Natl Acad Sci U S A , 110 :10177 , 2013
Abstract : Insect carboxylesterases from the alphaEsterase gene cluster, such as alphaE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcalphaE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of alpha-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcalphaE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical alpha/beta-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal alpha-helix that serves as a membrane anchor. Soaking of LcalphaE7 crystals in OPs led to the capture of a crystallographic snapshot of LcalphaE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcalphaE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants ( approximately 10(6) M(-1) s(-1)) indicative of a natural substrate.
ESTHER : Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
PubMedSearch : Jackson_2013_Proc.Natl.Acad.Sci.U.S.A_110_10177
PubMedID: 23733941
Gene_locus related to this paper: luccu-E3aest7

Title : Inhibition pathways of the potent organophosphate CBDP with cholinesterases revealed by X-ray crystallographic snapshots and mass spectrometry - Carletti_2013_Chem.Res.Toxicol_26_280
Author(s) : Carletti E , Colletier JP , Schopfer LM , Santoni G , Masson P , Lockridge O , Nachon F , Weik M
Ref : Chemical Research in Toxicology , 26 :280 , 2013
Abstract : Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 A snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.
ESTHER : Carletti_2013_Chem.Res.Toxicol_26_280
PubMedSearch : Carletti_2013_Chem.Res.Toxicol_26_280
PubMedID: 23339663
Gene_locus related to this paper: human-BCHE , mouse-ACHE

Title : Huprine derivatives as sub-nanomolar human acetylcholinesterase inhibitors: from rational design to validation by X-ray crystallography - Ronco_2012_ChemMedChem_7_400
Author(s) : Ronco C , Carletti E , Colletier JP , Weik M , Nachon F , Jean L , Renard PY
Ref : ChemMedChem , 7 :400 , 2012
Abstract : This complete study - from rational design to validation by X-ray crystallography allowed us to discover two sub-nanomolar hAChE inhibitors (430 and 530 pM) grafted with an easily derivatized linker directed toward the AChE peripheral site. The crystal structure of mouse AChE in complex with compound 4 was solved and confirms the favorable position of the triazole in the active site gorge, paving the way for a new class of bifunctional ligands.
ESTHER : Ronco_2012_ChemMedChem_7_400
PubMedSearch : Ronco_2012_ChemMedChem_7_400
PubMedID: 22052791
Gene_locus related to this paper: mouse-ACHE

Title : Energy landscapes of human acetylcholinesterase and its huperzine a-inhibited counterpart - Trapp_2012_J.Phys.Chem.B_116_14744
Author(s) : Trapp M , Trovaslet M , Nachon F , Koza MM , van Eijck L , Hill F , Weik M , Masson P , Tehei M , Peters J
Ref : J Phys Chem B , 116 :14744 , 2012
Abstract : Enzymes are animated by a hierarchy of motions occurring on time scales that span more than 15 orders of magnitude from femtoseconds (10(-15) s) to several minutes. As a consequence, an enzyme is characterized by a large number of conformations, so-called conformational substates that interconvert via molecular motions. The energy landscapes of these macromolecules are very complex, and many conformations are separated by only small energy barriers. Movements at this level are fast thermal atomic motions occurring on a time scale between 10(-7) and 10(-12) s, which are experimentally accessible by incoherent neutron scattering techniques. They correspond to local fluctuations within the molecule and are believed to act as coupling links for larger, conformational changes. Several questions related to this hierarchy of motions are a matter of very active research: which of the motions are involved in the biological functions of the macromolecule and are motions of different energy (and thus time) scale correlated? How does the distribution of motions change when an enzyme is inhibited? We report here on investigations of the enzyme human acetylcholinesterase, unliganded and in complex with the noncovalent inhibitor Huperzine A, by incoherent neutron scattering. Different time scales are explored to shed light on the interplay of enzyme activity, dynamics, and inhibition. Surprisingly the average molecular dynamics do not seem to be altered by the presence of the inhibitor used in this study within the considered time scales. The activation energy for the free and the inhibited form of the enzyme is moreover found to be almost identical despite changes of interactions inside the gorge, which leads to the active site of the enzyme.
ESTHER : Trapp_2012_J.Phys.Chem.B_116_14744
PubMedSearch : Trapp_2012_J.Phys.Chem.B_116_14744
PubMedID: 23186408

Title : Backdoor opening mechanism in acetylcholinesterase based on X-ray crystallography and molecular dynamics simulations - Sanson_2011_Protein.Sci_20_1114
Author(s) : Sanson B , Colletier JP , Xu Y , Lang PT , Jiang H , Silman I , Sussman JL , Weik M
Ref : Protein Science , 20 :1114 , 2011
Abstract : The transient opening of a backdoor in the active-site wall of acetylcholinesterase, one of nature's most rapid enzymes, has been suggested to contribute to the efficient traffic of substrates and products. A crystal structure of Torpedo californica acetylcholinesterase in complex with the peripheral-site inhibitor aflatoxin is now presented, in which a tyrosine at the bottom of the active-site gorge rotates to create a 3.4-A wide exit channel. Molecular dynamics simulations show that the opening can be further enlarged by movement of Trp84. The crystallographic and molecular dynamics simulation data thus point to the interface between Tyr442 and Trp84 as the key element of a backdoor, whose opening permits rapid clearance of catalysis products from the active site. Furthermore, the crystal structure presented provides a novel template for rational design of inhibitors and reactivators, including anti-Alzheimer drugs and antidotes against organophosphate poisoning.
ESTHER : Sanson_2011_Protein.Sci_20_1114
PubMedSearch : Sanson_2011_Protein.Sci_20_1114
PubMedID: 21594947
Gene_locus related to this paper: torca-ACHE

Title : Reaction of cresyl saligenin phosphate, the organophosphorus agent implicated in aerotoxic syndrome, with human cholinesterases: mechanistic studies employing kinetics, mass spectrometry, and X-ray structure analysis - Carletti_2011_Chem.Res.Toxicol_24_797
Author(s) : Carletti E , Schopfer LM , Colletier JP , Froment MT , Nachon F , Weik M , Lockridge O , Masson P
Ref : Chemical Research in Toxicology , 24 :797 , 2011
Abstract : Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k(i)), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k(i) of 1.6 x 10(8) M(-1) min(-1) and 2.7 x 10(7) M(-1) min(-1) for E and E' forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in (18)O-water showed that (18)O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.
ESTHER : Carletti_2011_Chem.Res.Toxicol_24_797
PubMedSearch : Carletti_2011_Chem.Res.Toxicol_24_797
PubMedID: 21438623
Gene_locus related to this paper: human-BCHE

Title : Long route or shortcut? A molecular dynamics study of traffic of thiocholine within the active-site gorge of acetylcholinesterase - Xu_2010_Biophys.J_99_4003
Author(s) : Xu Y , Colletier JP , Weik M , Qin G , Jiang H , Silman I , Sussman JL
Ref : Biophysical Journal , 99 :4003 , 2010
Abstract : The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.
ESTHER : Xu_2010_Biophys.J_99_4003
PubMedSearch : Xu_2010_Biophys.J_99_4003
PubMedID: 21156143

Title : Kinetic insight into the mechanism of cholinesterasterase inhibition by aflatoxin B1 to develop biosensors - Hansmann_2009_Biosens.Bioelectron_24_2119
Author(s) : Hansmann T , Sanson B , Stojan J , Weik M , Marty JL , Fournier D
Ref : Biosensors & Bioelectronics , 24 :2119 , 2009
Abstract : In this paper, the inhibition effect of aflatoxin B1 on different species of cholinesterases was investigated to unravel action mechanism. The inhibition curves of several cholinesterase mutants (obtained by spectrophotometric measurements of enzyme activity, pS curves) were analyzed. They showed that this toxin reversibly inhibits cholinesterases by binding to a peripheral site located at the entrance of the active site gorge without entering inside the site. Electric eel enzyme revealed the highest inhibition extent with a binding constant estimated to 0.35 microM. This binding prevents the entrance of substrate en route to the catalytic site and also decreases chemical steps of the reaction at the catalytic site: acetylation is reduced to the half and deacetylation is reduced to the third. Electric eel acetylcholinesterase was used to settle an amperometric biosensor. The best detection was obtained by using 0.3 mU enzyme on the electrode and 0.5mM ATCh in the solution. The limit of detection was 3 microM corresponding to 20% inhibition.
ESTHER : Hansmann_2009_Biosens.Bioelectron_24_2119
PubMedSearch : Hansmann_2009_Biosens.Bioelectron_24_2119
PubMedID: 19109006

Title : Crystallographic snapshots of nonaged and aged conjugates of soman with acetylcholinesterase, and of a ternary complex of the aged conjugate with pralidoxime - Sanson_2009_J.Med.Chem_52_7593
Author(s) : Sanson B , Nachon F , Colletier JP , Froment MT , Toker L , Greenblatt HM , Sussman JL , Ashani Y , Masson P , Silman I , Weik M
Ref : Journal of Medicinal Chemistry , 52 :7593 , 2009
Abstract : Organophosphate compounds (OP) are potent inhibitors of acetylcholinesterases (AChEs) and can cause lethal poisoning in humans. Inhibition of AChEs by the OP soman involves phosphonylation of the catalytic serine, and subsequent dealkylation produces a form known as the "aged" enzyme. The nonaged form can be reactivated to a certain extent by nucleophiles, such as pralidoxime (2-PAM), whereas aged forms of OP-inhibited AChEs are totally resistant to reactivation. Here, we solved the X-ray crystal structures of AChE from Torpedo californica (TcAChE) conjugated with soman before and after aging. The absolute configuration of the soman stereoisomer adduct in the nonaged conjugate is P(S)C(R). A structural reorientation of the catalytic His440 side chain was observed during the aging process. Furthermore, the crystal structure of the ternary complex of the aged conjugate with 2-PAM revealed that the orientation of the oxime function does not permit nucleophilic attack on the phosphorus atom, thus providing a plausible explanation for its failure to reactivate the aged soman/AChE conjugate. Together, these three crystal structures provide an experimental basis for the design of new reactivators.
ESTHER : Sanson_2009_J.Med.Chem_52_7593
PubMedSearch : Sanson_2009_J.Med.Chem_52_7593
PubMedID: 19642642
Gene_locus related to this paper: torca-ACHE

Title : Direct correlation between molecular dynamics and enzymatic stability: a comparative neutron scattering study of native human butyrylcholinesterase and its aged soman conjugate - Gabel_2009_Biophys.J_96_1489
Author(s) : Gabel F , Masson P , Froment MT , Doctor BP , Saxena A , Silman I , Zaccai G , Weik M
Ref : Biophysical Journal , 96 :1489 , 2009
Abstract : An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its "aged" soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.
ESTHER : Gabel_2009_Biophys.J_96_1489
PubMedSearch : Gabel_2009_Biophys.J_96_1489
PubMedID: 19217865

Title : Shoot-and-Trap: use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography - Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
Author(s) : Colletier JP , Bourgeois D , Sanson B , Fournier D , Sussman JL , Silman I , Weik M
Ref : Proc Natl Acad Sci U S A , 105 :11742 , 2008
Abstract : Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the "off-switch" at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a "backdoor." The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
ESTHER : Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
PubMedSearch : Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
PubMedID: 18701720
Gene_locus related to this paper: torca-ACHE

Title : Induced-fit or preexisting equilibrium dynamics? Lessons from protein crystallography and MD simulations on acetylcholinesterase and implications for structure-based drug design - Xu_2008_Protein.Sci_17_601
Author(s) : Xu Y , Colletier JP , Jiang H , Silman I , Sussman JL , Weik M
Ref : Protein Science , 17 :601 , 2008
Abstract : Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets.
ESTHER : Xu_2008_Protein.Sci_17_601
PubMedSearch : Xu_2008_Protein.Sci_17_601
PubMedID: 18359854

Title : Flexibility of aromatic residues in the active-site gorge of acetylcholinesterase: X-ray versus molecular dynamics - Xu_2008_Biophys.J_95_2500
Author(s) : Xu Y , Colletier JP , Weik M , Jiang H , Moult J , Silman I , Sussman JL
Ref : Biophysical Journal , 95 :2500 , 2008
Abstract : The high aromatic content of the deep and narrow active-site gorge of acetylcholinesterase (AChE) is a remarkable feature of this enzyme. Here, we analyze conformational flexibility of the side chains of the 14 conserved aromatic residues in the active-site gorge of Torpedo californica AChE based on the 47 three-dimensional crystal structures available for the native enzyme, and for its complexes and conjugates, and on a 20-ns molecular dynamics (MD) trajectory of the native enzyme. The degree of flexibility of these 14 aromatic side chains is diverse. Although the side-chain conformations of F330 and W279 are both very flexible, the side-chain conformations of F120, W233, W432, Y70, Y121, F288, F290 and F331 appear to be fixed. Residues located on, or adjacent to, the Omega-loop (C67-C94), namely W84, Y130, Y442, and Y334, display different flexibilities in the MD simulations and in the crystal structures. An important outcome of our study is that the majority of the side-chain conformations observed in the 47 Torpedo californica AChE crystal structures are faithfully reproduced by the MD simulation on the native enzyme. Thus, the protein can assume these conformations even in the absence of the ligand that permitted their experimental detection. These observations are pertinent to structure-based drug design.
ESTHER : Xu_2008_Biophys.J_95_2500
PubMedSearch : Xu_2008_Biophys.J_95_2500
PubMedID: 18502801

Title : Mechanisms of cholinesterase inhibition by inorganic mercury - Frasco_2007_FEBS.J_274_1849
Author(s) : Frasco MF , Colletier JP , Weik M , Carvalho F , Guilhermino L , Stojan J , Fournier D
Ref : Febs J , 274 :1849 , 2007
Abstract : The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.
ESTHER : Frasco_2007_FEBS.J_274_1849
PubMedSearch : Frasco_2007_FEBS.J_274_1849
PubMedID: 17355286
Gene_locus related to this paper: human-BCHE

Title : Use of a 'caged' analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography - Colletier_2007_Acta.Crystallogr.D.Biol.Crystallogr_63_1115
Author(s) : Colletier JP , Royant A , Specht A , Sanson B , Nachon F , Masson P , Zaccai G , Sussman JL , Goeldner M , Silman I , Bourgeois D , Weik M
Ref : Acta Crystallographica D Biol Crystallogr , 63 :1115 , 2007
Abstract : Acetylcholinesterase plays a crucial role in nerve-impulse transmission at cholinergic synapses. The apparent paradox that it displays high turnover despite its active site being buried raises cogent questions as to how the traffic of substrates and products to and from the active site can occur so rapidly in such circumstances. Here, a kinetic crystallography strategy aimed at structurally addressing the issue of product traffic in acetylcholinesterase is presented, in which UV-laser-induced cleavage of a photolabile precursor of the enzymatic product analogue arsenocholine, 'caged' arsenocholine, is performed in a temperature-controlled X-ray crystallography regime. The 'caged' arsenocholine was shown to bind at both the active and peripheral sites of acetylcholinesterase. UV irradiation of a complex with acetylcholinesterase during a brief temperature excursion from 100 K to room temperature is most likely to have resulted in a decrease in occupancy by the caged compound. Microspectrophotometric experiments showed that the caged compound had indeed been photocleaved. It is proposed that a fraction of the arsenocholine molecules released within the crystal had been expelled from both the active and the peripheral sites. Partial q-weighted difference refinement revealed a relative movement of the two domains in acetylcholinesterase after photolysis and the room-temperature excursion, resulting in an increase in the active-site gorge volume of 30% and 35% in monomers A and B of the asymmetric unit, respectively. Moreover, an alternative route to the active-site gorge of the enzyme appeared to open. This structural characterization of acetylcholinesterase 'at work' is consistent with the idea that choline exits from the enzyme after catalysis either via the gorge or via an alternative 'backdoor' trajectory.
ESTHER : Colletier_2007_Acta.Crystallogr.D.Biol.Crystallogr_63_1115
PubMedSearch : Colletier_2007_Acta.Crystallogr.D.Biol.Crystallogr_63_1115
PubMedID: 18007027
Gene_locus related to this paper: torca-ACHE

Title : [Exploring the conformational energy landscape of acetylcholinesterase by kinetic crystallography] - Colletier_2007_Ann.Pharm.Fr_65_108
Author(s) : Colletier JP , Weik M
Ref : Ann Pharm Fr , 65 :108 , 2007
Abstract : Acetylcholinesterase is a very rapid enzyme, essential in the process of nerve impulse transmission at cholinergic synapses. It is the target of all currently approved anti-Alzheimer drugs and further progress in the modulation of its activity requires structural as well as dynamical information. Exploration of the conformational energy landscape of a protein by means of X-ray crystallography requires the use of experimental tricks, to overcome the inherently static nature of crystallographic structures. Here we report three experimental approaches that allowed to gain structural insight into the dynamics of acetylcholinesterase, which is relevant for structure-based drug design.
ESTHER : Colletier_2007_Ann.Pharm.Fr_65_108
PubMedSearch : Colletier_2007_Ann.Pharm.Fr_65_108
PubMedID: 17404544

Title : Conformational flexibility in the peripheral site of Torpedo californica acetylcholinesterase revealed by the complex structure with a bifunctional inhibitor - Colletier_2006_J.Am.Chem.Soc_128_4526
Author(s) : Colletier JP , Sanson B , Nachon F , Gabellieri E , Fattorusso C , Campiani G , Weik M
Ref : Journal of the American Chemical Society , 128 :4526 , 2006
Abstract : The X-ray crystallographic structure of Torpedo californica acetylcholinesterase (TcAChE) in complex with the bifunctional inhibitor NF595, a potentially new anti-Alzheimer drug, has been solved. For the first time in TcAChE, a major conformational change in the peripheral-site tryptophan residue is observed upon complexation. The observed conformational flexibility highlights the dynamic nature of protein structures and is of importance for structure-based drug design.
ESTHER : Colletier_2006_J.Am.Chem.Soc_128_4526
PubMedSearch : Colletier_2006_J.Am.Chem.Soc_128_4526
PubMedID: 16594661
Gene_locus related to this paper: torca-ACHE

Title : Structural insights into substrate traffic and inhibition in acetylcholinesterase - Colletier_2006_EMBO.J_25_2746
Author(s) : Colletier JP , Fournier D , Greenblatt HM , Stojan J , Sussman JL , Zaccai G , Silman I , Weik M
Ref : EMBO Journal , 25 :2746 , 2006
Abstract : Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.
ESTHER : Colletier_2006_EMBO.J_25_2746
PubMedSearch : Colletier_2006_EMBO.J_25_2746
PubMedID: 16763558
Gene_locus related to this paper: torca-ACHE

Title : Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study - Gabel_2005_Biophys.J_89_3303
Author(s) : Gabel F , Weik M , Masson P , Renault F , Fournier D , Brochier L , Doctor BP , Saxena A , Silman I , Zaccai G
Ref : Biophysical Journal , 89 :3303 , 2005
Abstract : Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
ESTHER : Gabel_2005_Biophys.J_89_3303
PubMedSearch : Gabel_2005_Biophys.J_89_3303
PubMedID: 16100272

Title : Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals - Weik_2004_Biophys.J_86_3176
Author(s) : Weik M , Vernede X , Royant A , Bourgeois D
Ref : Biophysical Journal , 86 :3176 , 2004
Abstract : Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.
ESTHER : Weik_2004_Biophys.J_86_3176
PubMedSearch : Weik_2004_Biophys.J_86_3176
PubMedID: 15111430

Title : Poster (54) Experimental approaches to study the dynamics of cholinesterases. -
Author(s) : Weik M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :349 , 2004
PubMedID:

Title : Poster (74) Structural studies on torpedo californica acetylcholinesterase in complex with a substrate analogue -
Author(s) : Colletier JP , Fournier D , Greenblatt HM , Sussman JL , Zaccai G , Silman I , Weik M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :359 , 2004
PubMedID:

Title : The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study - Gabel_2004_Biophys.J_86_3152
Author(s) : Gabel F , Weik M , Doctor BP , Saxena A , Fournier D , Brochier L , Renault F , Masson P , Silman I , Zaccai G
Ref : Biophysical Journal , 86 :3152 , 2004
Abstract : A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
ESTHER : Gabel_2004_Biophys.J_86_3152
PubMedSearch : Gabel_2004_Biophys.J_86_3152
PubMedID: 15111428

Title : Evidence for the formation of disulfide radicals in protein crystals upon X-ray irradiation - Weik_2002_J.Synchrotron.Radiat_9_342
Author(s) : Weik M , Berges J , Raves ML , Gros P , McSweeney S , Silman I , Sussman JL , Houee-Levin C , Ravelli RB
Ref : J Synchrotron Radiat , 9 :342 , 2002
Abstract : Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by approximately 0.7 A upon X-ray irradiation as seen in a series of nine data sets collected on a single crystal. Simulation of the same bond suggests elongation by a similar value if a disulfide-radical anion is formed by trapping an electron. The absorption spectrum of a crystal irradiated under similar conditions shows a peak at approximately 400 nm, which in aqueous solution has been attributed to disulfide radicals. The results suggest that the formation of disulfide radicals in protein crystals owing to X-ray irradiation can be observed experimentally, both by structural means and by absorption spectroscopy.
ESTHER : Weik_2002_J.Synchrotron.Radiat_9_342
PubMedSearch : Weik_2002_J.Synchrotron.Radiat_9_342
PubMedID: 12409620

Title : Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes - Weik_2001_Protein.Sci_10_1953
Author(s) : Weik M , Ravelli RB , Silman I , Sussman JL , Gros P , Kroon J
Ref : Protein Science , 10 :1953 , 2001
Abstract : The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.
ESTHER : Weik_2001_Protein.Sci_10_1953
PubMedSearch : Weik_2001_Protein.Sci_10_1953
PubMedID: 11567086

Title : Specific chemical and structural damage to proteins produced by synchrotron radiation - Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
Author(s) : Weik M , Ravelli RB , Kryger G , McSweeney S , Raves ML , Harel M , Gros P , Silman I , Kroon J , Sussman JL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 97 :623 , 2000
Abstract : Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.
ESTHER : Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
PubMedSearch : Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
PubMedID: 10639129
Gene_locus related to this paper: torca-ACHE