A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

ATP binding and ATP hydrolysis in full-length MsbA monitored via time-resolved Fourier transform infrared spectroscopy.

The essential Escherichia coli ATPase MsbA is a lipid flippase that serves as a prototype for multi drug resistant ABC transporters. Its physiological function is the transport of lipopolisaccharides to build up the outer membranes of Gram-negative bacteria. Although several structural and biochemical studies of MsbA have been conducted previously, a detailed picture of the dynamic processes that link ATP hydrolysis to allocrit transport remains elusive. We report here for the first time time-resolved Fourier transform infrared (FTIR) spectroscopic measurements of the ATP binding and ATP hydrolysis reaction of full-length MsbA and determined reaction rates at 288 K of k 1 = 0.49 ± 0.28 s-1 and k 2 = 0.014 ± 0.003 s-1, respectively. We further verified these rates with photocaged NPEcgAppNHp where only nucleotide binding was observable and the negative mutant MsbA-H537A that showed slow hydrolysis (k 2 < 2 × 10-4 s-1). Besides single turnover kinetics, FTIR measurements also deliver IR signatures of all educts, products and the protein. ADP remains protein-bound after ATP hydrolysis. In addition, the spectral changes observed for the two variants MsbA-S378A and MsbA-S482A correlated with the loss of hydrogen bonding to the γ-phosphate of ATP. This study paves the way for FTIR-spectroscopic investigations of allocrite transport in full-length MsbA.

Microsecond-Resolved Infrared Spectroscopy on Nonrepetitive Protein Reactions by Applying Caged Compounds and Quantum Cascade Laser Frequency Combs.

Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about 10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of nonrepetitive reactions, e.g., induced by caged compounds, were limited to the millisecond time domain. The advent of dual-comb quantum cascade laser now allows for a rapid reaction monitoring in the microsecond time domain. Here, we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound-induced reactions monitored by FTIR and dual-comb spectroscopy by applying the new technique to the α subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gαi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with a 4 µs time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable microsecond time regime for nonrepetitive biological systems including all GTPases and ATPases.

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.

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases.

GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.

Mechanism of the intrinsic arginine finger in heterotrimeric G proteins.

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward ß-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

Monitoring transient events in infrared spectra using local mode analysis.

Time-resolved Fourier transformed infrared (FTIR) spectroscopy of chemical reactions is highly sensitive to minimal spatiotemporal changes. Structural features are decoded and represented in a comprehensible manner by combining FTIR spectroscopy with biomolecular simulations. Local mode analysis (LMA) is a tool to connect molecular motion based on a quantum mechanics simulation with infrared (IR) spectral features and vice versa. Here, we present the python-based software tool of LMA and demonstrate the novel feature of LMA to extract transient structural details and identify the related IR spectra at the case example of malonaldehyde (MA). Deuterated MA exists in two almost equally populated tautomeric states separated by a low barrier for proton transfer so IR spectra represent a mixture of both states. By state-dependent LMA, we obtain pure spectra for each tautomeric state occurring within the quantum mechanics trajectory. By time-resolved LMA, we obtain a clear view of the transition between states in the spectrum. Through local mode decomposition and the band-pass filter, marker bands for each state are identified. Thus, LMA is beneficial to analyze the experimental spectra based on a mixture of states by determining the individual contributions to the spectrum and motion of each state.

Label-free identification of myopathological features with coherent anti-Stokes Raman scattering.

INTRODUCTION: The aim of this study was the label-free identification of distinct myopathological features with coherent anti-Stokes Raman scattering (CARS) imaging, which leaves the sample intact for further analysis. METHODS: The protein distribution was determined without labels by CARS at 2,930 cm-1 and was compared with the results of standard histological staining. RESULTS: CARS imaging allowed the visualization of glycogen accumulation in glycogen storage disease type 5 (McArdle disease) and of internal nuclei in centronuclear myopathy. CARS identified an inhomogeneous protein distribution within muscle fibers in sporadic inclusion body myositis that was not shown with standard staining. In Duchenne muscular dystrophy, evidence for a higher protein content at the border of hypercontracted fibers was detected. DISCUSSION: CARS enables the label-free identification of distinct myopathological features, possibly paving the way for subsequent proteomic, metabolic, and genomic analyses. Muscle Nerve 58: 457-460, 2018.

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.

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.

Raman Microspectroscopic Evidence for the Metabolism of a Tyrosine Kinase Inhibitor, Neratinib, in Cancer Cells.

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.

Elucidation of Single Hydrogen Bonds in GTPases via Experimental and Theoretical Infrared Spectroscopy.

Time-resolved Fourier transform infrared (FTIR) spectroscopy is a powerful tool to elucidate label-free the reaction mechanisms of proteins. After assignment of the absorption bands to individual groups of the protein, the order of events during the reaction mechanism can be monitored and rate constants can be obtained. Additionally, structural information is encoded into infrared spectra and can be decoded by combining the experimental data with biomolecular simulations. We have determined recently the infrared vibrations of GTP and guanosine diphosphate (GDP) bound to Gαi1, a ubiquitous GTPase. These vibrations are highly sensitive for the environment of the phosphate groups and thereby for the binding mode the GTPase adopts to enable fast hydrolysis of GTP. In this study we calculated these infrared vibrations from biomolecular simulations to transfer the spectral information into a computational model that provides structural information far beyond crystal structure resolution. Conformational ensembles were generated using 15 snapshots of several 100 ns molecular-mechanics/molecular-dynamics (MM-MD) simulations, followed by quantum-mechanics/molecular-mechanics (QM/MM) minimization and normal mode analysis. In comparison with other approaches, no time-consuming QM/MM-MD simulation was necessary. We carefully benchmarked the simulation systems by deletion of single hydrogen bonds between the GTPase and GTP through several Gαi1 point mutants. The missing hydrogen bonds lead to blue-shifts of the corresponding absorption bands. These band shifts for α-GTP (Gαi1-T48A), γ-GTP (Gαi1-R178S), and for both ß-GTP/γ-GTP (Gαi1-K46A, Gαi1-D200E) were found in agreement in the experimental and the theoretical spectra. We applied our approach to open questions regarding Gαi1: we show that the GDP state of Gαi1 carries a Mg2+, which is not found in x-ray structures. Further, the catalytic role of K46, a central residue of the P-loop, and the protonation state of the GTP are elucidated.

Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs.

GTPases are central switches in cells. Their dysfunctions are involved in severe diseases. The small GTPase Ras regulates cell growth, differentiation and apoptosis by transmitting external signals to the nucleus. In one group of oncogenic mutations, the 'switch-off' reaction is inhibited, leading to persistent activation of the signaling pathway. The switch reaction is regulated by GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis in Ras, and by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. Heterotrimeric G-proteins are activated by G-protein coupled receptors and are inactivated by GTP hydrolysis in the Gα subunit. Their GAPs are called regulators of G-protein signaling. In the same way that Ras serves as a prototype for small GTPases, Gαi1 is the most well-studied Gα subunit. By utilizing X-ray structural models, time-resolved infrared-difference spectroscopy, and biomolecular simulations, we elucidated the detailed molecular reaction mechanism of the GTP hydrolysis in Ras and Gαi1. In both proteins, the charge distribution of GTP is driven towards the transition state, and an arginine is precisely positioned to facilitate nucleophilic attack of water. In addition to these mechanistic details of GTP hydrolysis, Ras dimerization as an emerging factor in signal transduction is discussed in this review.

Unraveling the Phosphocholination Mechanism of the Legionella pneumophila Enzyme AnkX.

The intracellular pathogen Legionella pneumophila 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.

Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism.

Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg(2+) binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s(-1) at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, ß-, and γ-GTP (1243, 1224, and 1156 cm(-1), respectively), α- and ß-GDP (1214 and 1134/1103 cm(-1), respectively), and free phosphate (1078/991 cm(-1)). In contrast to Ras · GAP catalysis, the bond breakage of the ß-γ-phosphate but not the Pi release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like/all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated.

Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy.

Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis.

Amyloid-ß-Secondary Structure Distribution in Cerebrospinal Fluid and Blood Measured by an Immuno-Infrared-Sensor: A Biomarker Candidate for Alzheimer's Disease.

The misfolding of the Amyloid-beta (Aß) peptide into ß-sheet enriched conformations was proposed as an early event in Alzheimer's Disease (AD). Here, the Aß peptide secondary structure distribution in cerebrospinal fluid (CSF) and blood plasma of 141 patients was measured with an immuno-infrared-sensor. The sensor detected the amide I band, which reflects the overall secondary structure distribution of all Aß peptides extracted from the body fluid. We observed a significant downshift of the amide I band frequency of Aß peptides in Dementia Alzheimer type (DAT) patients, which indicated an overall shift to ß-sheet. The secondary structure distribution of all Aß peptides provides a better marker for DAT detection than a single Aß misfold or the concentration of a specific oligomer. The discrimination between DAT and disease control patients according to the amide I frequency was in excellent agreement with the clinical diagnosis (accuracy 90% for CSF and 84% for blood). The amide I band maximum above or below the decisive marker frequency appears as a novel spectral biomarker candidate of AD. Additionally, a preliminary proof-of-concept study indicated an amide I band shift below the marker band already in patients with mild cognitive impairment due to AD. The presented immuno-IR-sensor method represents a promising, simple, robust, and label-free diagnostic tool for CSF and blood analysis.

Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy.

Membrane trafficking is regulated by small Ras-like GDP/GTP binding proteins of the Rab subfamily (Rab GTPases) that cycle between membranes and cytosol depending on their nucleotide state. The GDP dissociation inhibitor (GDI) solubilizes prenylated Rab GTPases from and shuttles them between membranes in the form of a soluble cytosolic complex. We use attenuated total reflection-Fourier transform infrared spectroscopy to directly observe extraction of Rab GTPases from model membranes by GDI. In their native form, most Rab GTPases are doubly geranylgeranylated at the C terminus to achieve localization to the membrane. We find that monogeranylgeranylated Rab35 and Rab1b reversibly bind to a negatively charged model membrane. Correct folding and GTPase activity of the membrane-bound protein can be evaluated. The dissociation kinetics depends on the C-terminal sequence and charge of the GTPases. The attenuated total reflection experiments show that GDI genuinely accelerates the intrinsic Rab membrane dissociation. The extraction process is characterized and occurs in a nucleotide-dependent manner. Furthermore, we find that phosphocholination of Rab35, which is catalyzed by the Legionella pneumophila protein AnkX, interferes with the ability of GDI to extract Rab35 from the membrane. The attenuated total reflection-Fourier transform infrared spectroscopy approach enables label-free investigation of the interaction between GDI and Rab GTPases in a membrane environment. Thereby, GDI is revealed to actively extract monogeranylgeranylated membrane-bound Rab GTPases and, thus, is not merely a solubilization factor.

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.

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

Predictions about the cellular efficacy of drugs tested in vitro 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 in vivo cellular response to drugs. This might explain some large discrepancies between in vitro drug studies and drug responses observed in patients. We present a novel in vitro 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 in vitro. The Raman studies detect large panitumumab-induced differences in vitro 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, in vitro 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.