Detailed Structure of the H₂PO₄^––Guanosine Diphosphate Intermediate in Ras-GAP Decoded from FTIR Experiments by Biomolecular Simulations

Essential biochemical processes such as signal transduction, energy conversion, or substrate conversion depend on transient ligand binding. Thus, identifying the detailed structure and transient positioning of small ligands, and their stabilization by the surrounding protein, is of great importance. In this study, by decoding information from Fourier transform infrared (FTIR) spectra with biomolecular simulation methods, we identify the precise position and hydrogen network of a small compound, the guanosine diphosphate (GDP)–H₂PO₄^– intermediate, in the surrounding protein–protein complex of Ras and its GTPase-activating protein, a central molecular switch in cellular signal transduction. We validate the simulated structure by comparing the calculated fingerprint vibrational modes of H₂PO₄^– with those obtained from FTIR experiments. The new structural information, below the resolution of X-ray structural analysis, gives detailed insight into the catalytic mechanism

Exploring the Multidimensional Free Energy Surface of Phosphoester Hydrolysis with Constrained QM/MM Dynamics

The mechanism of the hydrolysis of phosphate monoesters, a ubiquitous biological reaction, has remained under debate. We here investigated the hydrolysis of a nonenzymatic model system, the monomethyl phosphate dianion, by hybrid quantum mechanical and molecular mechanical simulations. The solvation effects were taken into account with explicit water. Detailed free energy landscapes in two-dimensional and three-dimensional space were resolved using the multidimensional potential of mean constraint force, a newly developed method that was demonstrated to be powerful for free energy calculations along multiple coordinates. As in previous theoretical studies, the associative and dissociative pathways were indistinguishable. Furthermore, the associative pathway was investigated in great detail. We propose a rotation of an O–H bond in the transition between two pentacoordinated structures, during which an overall transition state was identified with an activation energy of 50 kcal/mol. This is consistent with experimental data. The results support a concerted proton transfer from the nucleophilic water to the phosphate group, and then to the leaving group

Reaction Mechanism of Adenylyltransferase DrrA from <i>Legionella pneumophila</i> Elucidated by Time-Resolved Fourier Transform Infrared Spectroscopy

Modulation of the function of small GTPases that regulate vesicular trafficking is a strategy employed by several human pathogens. <i>Legionella pneumophila</i> infects lung macrophages and injects a plethora of different proteins into its host cell. Among these is DrrA/SidM, which catalyzes stable adenylylation of Rab1b, a regulator of endoplasmatic reticulum to Golgi trafficking, and thereby alters the function and interactions of this small GTPase. We employed time-resolved FTIR-spectroscopy to monitor the DrrA-catalyzed AMP-transfer to Tyr77 of Rab1b. A transient complex between DrrA, adenylylated Rab1b, and the pyrophosphate byproduct was resolved, allowing us to analyze the interactions at the active site. Combination of isotopic labeling and site-directed mutagenesis allowed us to derive the catalytic mechanism of DrrA from the FTIR difference spectra. DrrA shares crucial residues in the ATP-binding pocket with similar AMP-transferring enzymes such as glutamine synthetase adenylyltransferase or kanamycin nucleotidyltransferase, but provides the complete active site on a single subunit. We determined that Asp112 of DrrA functions as the catalytic base for deprotonation of Tyr77 of Rab1b to enable nucleophilic attack on the ATP. The study provides detailed understanding of the <i>Legionella pneumophila</i> protein DrrA and of AMP-transfer reactions in general

Universal Method for Protein Immobilization on Chemically Functionalized Germanium Investigated by ATR-FTIR Difference Spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy allows a detailed analysis of surface attached molecules, including their secondary structure, orientation, and interaction with small molecules in the case of proteins. Here, we present a universal immobilization technique on germanium for all oligo-histidine-tagged proteins. For this purpose, new triethoxysilane derivates were developed: we synthesized a linker–silane with a succinimidyl ester as amine-reactive headgroup and a matrix–silane with an unreactive ethylene glycol group. A new methodology for the attachment of triethoxysilanes on germanium was established, and the surface was characterized by ATR-FTIR and X-ray photoelectron spectroscopy. In the next step, the succinimidyl ester was reacted with aminonitrilotriacetic acid. Subsequently, Ni²⁺ was coordinated to form Ni–nitrilotriacetic acid for His-tag binding. The capability of the functionalized surface was demonstrated by experiments using the small GTPase Ras and photosystem I (PS I). The native binding of the proteins was proven by difference spectroscopy, which probes protein function. The function of Ras as molecular switch was demonstrated by a beryllium trifluoride anion titration assay, which allows observation of the “on” and “off” switching of Ras at atomic resolution. Furthermore, the activity of immobilized PS I was proven by light-induced difference spectroscopy. Subsequent treatment with imidazole removes attached proteins, enabling repeated binding. This universal technique allows specific attachment of His-tagged proteins and a detailed study of their function at the atomic level using FTIR difference spectroscopy

An ATR–FTIR Sensor Unraveling the Drug Intervention of Methylene Blue, Congo Red, and Berberine on Human Tau and Aβ

Alzheimer’s disease affects millions of human beings worldwide. The disease progression is characterized by the formation of plaques and neurofibrillary tangles in the brain, which are based on aggregation processes of the Aβ peptide and tau protein. Today there is no cure and even no <i>in vitro</i> assay available for the identification of drug candidates, which provides direct information concerning the protein secondary structure label-free. Therefore, we developed an attenuated total reflection Fourier transform infrared spectroscopy (ATR–FTIR) sensor, which uses surface bound antibodies to immobilize a desired target protein. The secondary structure of the protein can be evaluated based on the secondary structure sensitive frequency of the amide I band. Direct information about the effect of a drug candidate on the secondary structure distribution of the total target protein fraction within the respective body fluid can be detected by a frequency shift of the amide I band. Thereby, the extent of the amide I shift is indicative for the compound efficiency. The functionality of this approach was demonstrated by the quantification of the effect of the drug candidate methylene blue on the pathogenic misfolded tau protein as extracted from cerebrospinal fluid (CSF). Methylene blue induces a shift from pathogenic folded β-sheet dominated to the healthy monomeric state. A similar effect was observed for congo red on pathogenic Aβ isoforms from CSF. In addition, the effect of berberine on synthetic Aβ_1–42 is studied. Berberine seems to decelerate the aggregation process of synthetic Aβ_1–42 peptides

Specific Substates of Ras To Interact with GAPs and Effectors: Revealed by Theoretical Simulations and FTIR Experiments

The oncogenic Ras protein adopts various specific conformational states to execute its function in signal transduction. The large number of Ras structures obtained from X-ray and NMR experiments illustrates the diverse conformations that Ras adopts. It is difficult, however, to connect specific structural features with Ras functions. We report the free-energy landscape of Ras·GTP based on extensive explicit solvent simulations. The free-energy map clearly shows that the functional state 2 of Ras·GTP in fact has two distinct substates, denoted here as “Tyr32_in” and “Tyr32_out”. Unbiased MD simulations show that the two substrates interconvert on the submicrosecond scale in solution, pointing to a novel mechanism for Ras·GTP to selectively interact with GAPs and effectors. This proposal is further supported by time-resolved FTIR experiments, which demonstrate that Tyr32 destabilizes the Ras·GAP complex and facilitates an efficient termination of Ras signaling

Boletín de Segovia: Número 115 - 1910 septiembre 26

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Unraveling the Phosphocholination Mechanism of the <i>Legionella pneumophila</i> Enzyme AnkX

The intracellular pathogen <i>Legionella pneumophila</i> infects lung macrophages and injects numerous effector proteins into the host cell to establish a vacuole for proliferation. The necessary interference with vesicular trafficking of the host is achieved by modulation of the function of Rab GTPases. The effector protein AnkX chemically modifies Rab1b and Rab35 by covalent phosphocholination of serine or threonine residues using CDP-choline as a donor. So far, the phosphoryl transfer mechanism and the relevance of observed autophosphocholination of AnkX remained disputable. We designed tailored caged compounds to make this type of enzymatic reaction accessible for time-resolved Fourier transform infrared difference spectroscopy. By combining spectroscopic and biochemical methods, we determined that full length AnkX is autophosphocholinated at Ser521, Thr620, and Thr943. However, autophosphocholination loses specificity for these sites in shortened constructs and does not appear to be relevant for the catalysis of the phosphoryl transfer. In contrast, transient phosphocholination of His229 in the conserved catalytic motif might exist as a short-lived reaction intermediate. Upon substrate binding, His229 is deprotonated and locked in this state, being rendered capable of a nucleophilic attack on the pyrophosphate moiety of the substrate. The proton that originated from His229 is transferred to a nearby carboxylic acid residue. Thus, our combined findings support a ping-pong mechanism involving phosphocholination of His229 and subsequent transfer of phosphocholine to the Rab GTPase. Our approach can be extended to the investigation of further nucleotidyl transfer reactions, which are currently of reemerging interest in regulatory pathways of host–pathogen interactions

Label-Free Raman Spectroscopic Imaging Monitors the Integral Physiologically Relevant Drug Responses in Cancer Cells

Predictions about the cellular efficacy of drugs tested <i>in vitro</i> are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the <i>in vivo</i> cellular response to drugs. This might explain some large discrepancies between <i>in vitro</i> drug studies and drug responses observed in patients. We present a novel <i>in vitro</i> marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed <i>in vitro</i>. The Raman studies detect large panitumumab-induced differences <i>in vitro</i> in cells harboring wild-type K-Ras as seen in A in red but not in cells with K-Ras mutations as seen in B; these studies reflect the observed patient outcomes. However, the effect is not observed when extracellular-signal-regulated kinase phosphorylation is monitored. The Raman spectra show for cells with wild-type K-Ras alterations based on the responses to panitumumab. The subcellular component with the largest spectral response to panitumumab was lipid droplets, but this effect was not observed when cells harbored K-Ras mutations. This study develops a noninvasive, label-free, <i>in vitro</i> vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines. This approach opens a new field of patient-centered drug testing that could deliver superior patient therapies

Additional file 3 of Fully automated registration of vibrational microspectroscopic images in histologically stained tissue sections

Tissue Microarray Registration Results. Detailed registration results of 56 FTIR vs. H&E TMA cores. (PDF 46387.2 kb