The Ras dimer structure.

Oncogenic mutated Ras is a key player in cancer, but despite intense and expensive approaches its catalytic center seems undruggable. The Ras dimer interface is a possible alternative drug target. Dimerization at the membrane affects cell growth signal transduction. In vivo studies indicate that preventing dimerization of oncogenic mutated Ras inhibits uncontrolled cell growth. Conventional computational drug-screening approaches require a precise atomic dimer model as input to successfully access drug candidates. However, the proposed dimer structural models are controversial. Here, we provide a clear-cut experimentally validated N-Ras dimer structural model. We incorporated unnatural amino acids into Ras to enable the binding of labels at multiple positions via click chemistry. This labeling allowed the determination of multiple distances of the membrane-bound Ras-dimer measured by fluorescence and electron paramagnetic resonance spectroscopy. In combination with protein-protein docking and biomolecular simulations, we identified key residues for dimerization. Site-directed mutations of these residues prevent dimer formation in our experiments, proving our dimer model to be correct. The presented dimer structure enables computational drug-screening studies exploiting the Ras dimer interface as an alternative drug target.

Estimation of Protein-Ligand Unbinding Kinetics Using Non-Equilibrium Targeted Molecular Dynamics Simulations.

We here report on nonequilibrium targeted molecular dynamics simulations as a tool for the estimation of protein-ligand unbinding kinetics. Correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90, we show that the mean nonequilibrium work computed in an ensemble of trajectories of enforced ligand unbinding is a promising predictor for ligand unbinding rates. We furthermore investigate the molecular basis determining unbinding rates within the compound libraries. We propose ligand conformational changes and protein-ligand nonbonded interactions to impact on unbinding rates. Ligands may remain longer at the protein if they exhibit strong electrostatic and/or van der Waals interactions with the target. In the case of ligands with a rigid chemical scaffold that exhibit longer residence times, transient electrostatic interactions with the protein appear to facilitate unbinding. Our results imply that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding kinetics.

Ligand-Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free-Towards a Conformational Activity Screening for Drug Discovery.

Investigation of protein-ligand interactions is crucial during early drug-discovery processes. ATR-FTIR spectroscopy can detect label-free protein-ligand interactions with high spatiotemporal resolution. Here we immobilized, as an example, the heat shock protein HSP90 on an ATR crystal. This protein is an important molecular target for drugs against several diseases including cancer. With our novel approach we investigated a ligand-induced secondary structural change. Two specific binding modes of 19 drug-like compounds were analyzed. Different binding modes can lead to different efficacy and specificity of different drugs. In addition, the kobs values of ligand dissociation were obtained. The results were validated by X-ray crystallography for the structural change and by SPR experiments for the dissociation kinetics, but our method yields all data in a single and simple experiment.

Highly stable protein immobilization via maleimido-thiol chemistry to monitor enzymatic activity.

Immobilizing enzymes for biocatalysis offers many advantages, including easy separation of the enzyme from the product and repeated and continuous use. ATR-FTIR spectroscopy is a versatile tool to monitor immobilized enzymes and has been applied to many proteins. However, while the common and convenient immobilization via oligohistidine on mono-NTA layers is adequate for the measurement of difference spectra induced by ligand binding or photochemistry, it lacks the long term stability that is necessary for monitoring biocatalysis. Here, we report a new immobilization methodology based on maleimido-thiol chemistry. A 12-mercaptododecanoic acid NHS ester monolayer is reacted with 1-(2-aminoethyl)-maleimide to build a thiol reactive surface. Subsequently, NTA-C16-thiol is covalently attached and finally oligohistidine tagged enzymes were immobilized to this surface, which remained bound with a five times higher EC50-value compared to typical mono-NTA layers. To demonstrate the high potential of the surface we analysed decarboxylation reactions catalyzed by arylmalonate decarboxylase. With ATR-FTIR both the enzyme and its substrate conversion can be monitored label free. Correct folding of the enzyme can be evaluated based on the amide band of the immobilized enzyme. In addition, the infrared absorption spectra of educt and product are monitored in real time. We show that hybrid hard-soft multivariate curve resolution improves separation of the product and educt spectra from other effects during the experiments, leading to clean kinetic traces and reaction rates for the catalytic process. Our approach can in principle be extended to any enzyme and is ideally suited for the development of biocatalysts.

Amyloid blood biomarker detects Alzheimer's disease.

Alzheimer's disease (AD) is currently incurable, but there is general agreement that a minimally invasive blood biomarker for screening in preclinical stages would be crucial for future therapy. Diagnostic tools for detection of AD are either invasive like cerebrospinal fluid (CSF) biomarkers or expensive such as positron emission tomography (PET) scanning. Here, we determine the secondary structure change of amyloid-ß (Aß) in human blood. This change used as blood amyloid biomarker indicates prodromal AD and correlates with CSF AD biomarkers and amyloid PET imaging in the cross-sectional BioFINDER cohort. In a further population-based longitudinal cohort (ESTHER), the blood biomarker detected AD several years before clinical diagnosis in baseline samples with a positive likelihood ratio of 7.9; that is, those who were diagnosed with AD over the years were 7.9 times more likely to test positive. This assay may open avenues for blood screening of early AD stages as a funnel for further more invasive and expensive tests.

The protonation states of GTP and GppNHp in Ras proteins.

The small GTPase Ras transmits signals in a variety of cellular signaling pathways, most prominently in cell proliferation. GTP hydrolysis in the active center of Ras acts as a prototype for many GTPases and is the key to the understanding of several diseases, including cancer. Therefore, Ras has been the focus of intense research over the last decades. A recent neutron diffraction crystal structure of Ras indicated a protonated γ-guanylyl imidodiphosphate (γ-GppNHp) group, which has put the protonation state of GTP in question. A possible protonation of GTP was not considered in previously published mechanistic studies. To determine the detailed prehydrolysis state of Ras, we calculated infrared and NMR spectra from quantum mechanics/molecular mechanics (QM/MM) simulations and compared them with those from previous studies. Furthermore, we measured infrared spectra of GTP and several GTP analogs bound to lipidated Ras on a membrane system under near-native conditions. Our findings unify results from previous studies and indicate a structural model confirming the hypothesis that γ-GTP is fully deprotonated in the prehydrolysis state of Ras.

Asymmetric rhenium tricarbonyl complexes show superior luminescence properties in live cell imaging.

The synthesis and photophysical properties of a novel series of rhenium tricarbonyl complexes based on tridentate phenanthridinyl-containing ligands are described. Photophysical data reveal beneficial luminescence behaviour especially for compounds with an asymmetric ligand set. These advantageous properties are not limited to organic solvents, but indeed also improved in aqueous solutions. The suitability of our new rhenium complexes as potent imaging agents has been confirmed by fluorescence microscopy on living cancer cells, which also confirms superior long-time stability under fluorescence microscopy conditions. Colocalisation studies with commercial organelle stains reveal an accumulation of the complexes in the endoplasmic reticulum for all tested cell lines.

[FeFe]-Hydrogenase with Chalcogenide Substitutions at the H-Cluster Maintains Full H2 Evolution Activity.

The [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H-cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H-cluster into the HYDA1-apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4FeH ] cluster with selenium. The crystal structure of the reconstituted pre-mature HYDA1[4Fe4Se]H protein was determined, and a catalytically intact artificial H-cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native-like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X-ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity.

Chemical Functionalization of Germanium with Dextran Brushes for Immobilization of Proteins Revealed by Attenuated Total Reflection Fourier Transform Infrared Difference Spectroscopy.

Protein immobilization studied by attenuated total reflection Fourier transform infrared (ATR-FT-IR) difference spectroscopy is an emerging field enabling the study of proteins at atomic detail. Gold or glass surfaces are frequently used for protein immobilization. Here, we present an alternative method for protein immobilization on germanium. Because of its high refractive index and broad spectral window germanium is the best material for ATR-FT-IR spectroscopy of thin layers. So far, this technique was mainly used for protein monolayers, which lead to a limited signal-to-noise ratio. Further, undesired protein-protein interactions can occur in a dense layer. Here, the germanium surface was functionalized with thiols and stepwise a dextran brush was generated. Each step was monitored by ATR-FT-IR spectroscopy. We compared a 70 kDa dextran with a 500 kDa dextran regarding the binding properties. All surfaces were characterized by atomic force microscopy, revealing thicknesses between 40 and 110 nm. To analyze the capability of our system we utilized N-Ras on mono-NTA (nitrilotriacetic acid) functionalized dextran, and the amount of immobilized Ras corresponded to several monolayers. The protein stability and loading capacity was further improved by means of tris-NTA for immobilization. Small-molecule-induced changes were revealed with an over 3 times higher signal-to-noise ratio compared to monolayers. This improvement may allow the observation of very small and so far hidden changes in proteins upon stimulus. Furthermore, we immobilized green fluorescent protein (GFP) and mCherry simultaneously enabling an analysis of the surface by fluorescence microscopy. The absence of a Förster resonance energy transfer (FRET) signal demonstrated a large protein-protein distance, indicating an even distribution of the protein within the dextran.

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, Ni(2+) 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.

N-Ras forms dimers at POPC membranes.

Ras is a central regulator of cellular signaling pathways. It is mutated in 20-30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between ß2 and ß3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.

Surface-attached polyhistidine-tag proteins characterized by FTIR difference spectroscopy.

A universal label-free method for the spectroscopic investigation of polyhistidine-tagged proteins is presented. A solid supported lipid bilayer (SSLB, picture) containing nitrilotriacetic-acid-modified lipids is attached on top of a germanium attenuated total reflection crystal by hydrophilic interactions. Any His tag-modified protein can be immobilized and investigated by FTIR spectroscopy.

Secondary structure of lipidated Ras bound to a lipid bilayer.

Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post-translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance-FTIR studies of lipid-modified membrane-bound N-Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N-Ras was prepared by linking a farnesylated and hexadecylated N-Ras lipopeptide to a truncated N-Ras protein (residues 1-181). It was then bound to a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three-dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane-bound lipidated Ras, and provided a reliable decomposition for the membrane-bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane-bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G-domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane-bound Ras protein.

Sulfate acts as phosphate analog on the monomeric catalytic fragment of the CPx-ATPase CopB from Sulfolobus solfataricus.

The crystal structure of the catalytic fragment of a Sulfolobus solfataricus P-type ATPase, CopB-B, was determined with a 2.6 A resolution. CopB-B is the major soluble fragment of the archaeal CPx-ATPase CopB and is comprized of a nucleotide and a phosphorylation domain. In the crystalline state two molecules of CopB-B are in close contact to each other, although the presence of dimers in free solution could be ruled out by analytical ultracentrifugation. The overall architecture of CopB-B is similar to that of other P-type ATPases such as Ca-ATPase. Short peptide segments are linking the nucleotide binding to the phosphorylation domain. CopB-B exhibits 33% sequence identity (of 216 aligned residues) with the respective fragment of the Archaeoglobus fulgidus ATPase CopA. The CopB-B nucleotide-binding domain has the most primitive fold yet identified for this enzyme class. It is 24% identical to the nucleotide-binding domain of the disease-related Wilson ATPase ATP7B (80 structurally aligned residues). Structural superposition with Ca-ATPase suggests a putative nucleotide-binding site in CopB-B. The phosphorylation domain of CopB-B is structurally related to the corresponding part of Ca-ATPase in the anion-bound E2 state. In CopB-B crystals, a bound sulfate anion was identified at the phosphate-binding location. In solution state, the potential binding of CopB-B to phosphate was probed with (32)P(i). Bound phosphate could be readily displaced by orthovanadate at submillimolar concentration as well as by sulfate at millimolar concentration. It is possible therefore to assign the structure of the sulfate-bound phosphorylation domain of CopB-B to a state related to the E2.P(i) intermediate state of the catalytic cycle.