Maarten Merkx

Dioxygen Activation and Methane Hydroxylation by Soluble Methane Monooxygenase: A Tale of Two Irons and Three Proteins

Methanotrophic bacteria are capable of using methane as their sole source of carbon and energy. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by a fascinating enzyme system called methane monooxygenase (MMO). The selective oxidation of the very stable C-H bond in methane under ambient conditions is a remarkable feat that has not yet been repeated by synthetic catalysts and has attracted considerable scientific and commercial interest. The best studied MMO is a complex enzyme system that consists of three soluble protein components, all of which are required for efficient catalysis. Dioxygen activation and subsequent methane hydroxylation are catalyzed by a hydroxylase enzyme that contains a non-heme diiron site. A reductase protein accepts electrons from NADH and transfers them to the hydroxylase where they are used for the reductive activation of O2 . The third protein component couples electron and dioxygen consumption with methane oxidation. In this review we examine different aspects of catalysis by the MMO proteins, including the mechanisms of dioxygen activation at the diiron site and substrate hydroxylation by the activated oxygen species. We also discuss the role of complex formation between the different protein components in regulating various aspects of catalysis.

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic β cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles.

Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2-vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2-vsvG. Furthermore, hCTR2-vsvG was co-immunoprecipitated with hCTR2-FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2-eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)-luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE-luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 muM CuCl2, consistent with the K(m) of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-luciferase reporter activation in a copper-dependent manner at 40 and 100 microM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.

Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation

The key pathogenic event in the onset of Alzheimers disease (AD) is the aggregation of β‐amyloid (Aβ) peptides into toxic aggregates. Molecules that interfere with this process might act as therapeutic agents for the treatment of AD. The amino acid residues 16–20 (KLVFF) are known to be essential for the aggregation of Aβ. In this study, we have used a first‐generation dendrimer as a scaffold for the multivalent display of the KLVFF peptide. The effect of four KLVFF peptides attached to the dendrimer (K4) on Aβ aggregation was compared to the effect of monomeric KLVFF (K1). Our data show that K4 very effectively inhibits the aggregation of low‐molecular‐weight and protofibrillar Aβ1–42 into fibrils, in a concentration‐dependent manner, and much more potently than K1. Moreover, we show that K4 can lead to the disassembly of existing aggregates. Our data lead us to propose that conjugates that bear multiple copies of KLVFF might be useful as therapeutic agents for the treatment of Alzheimers disease.

Enhanced sensitivity of FRET-based protease sensors by redesign of the GFP dimerization interface

changes in a sensor domain are translated into a change in energy-transfer efficiency between donor and acceptor fluorescent domains, which is detected by a change in the ratio of donor and acceptor emission. This ratiometric response is independent of the sensor concentration, which is an important advantage of FRET-based sensors. In practice, however, most FRET-based sensors display only a relatively small difference in emission ratio upon activation. Improvement of these ratiometric changes has been recognized as an important prerequisite for use of these sensor systems in high-throughput applications based on fluorescence plate readers and fluorescence assisted cell sorting (FACS). [14, 15] Recently a pair of CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) variants, CyPet and YPet, respectively, have been reported that were optimized for FRET through a process of directed evolution. [16] When incorporated in a protease sensor, a 20-fold change in emission ratio was observed upon cleavage of a flexible peptide that linked CyPet and YPet, compared to only a fourfold change for the same construct with enhanced CFP (ECFP) and enhanced YFP (EYFP) domains. However, the mechanism behind their remarkable FRET properties has remained unclear. A total of eighteen mutations were introduced in the course of their development, many of which were at the exterior of the protein, at a large distance from the fluorophore. Moreover, no large differences in quantum yield or extinction coefficient were reported; this suggests that the photophysical properties of the fluorescent proteins were not significantly altered. We therefore hypothesized that the increase in FRET observed for CyPet and YPet could be due to an enhanced tendency to interact when connected by a peptide linker. The parent green fluorescent protein (GFP) has a known tendency to dimerize, [17] and analysis of the mutations in YPet have identified two residues, S208F and V224L, that are present at the dimer interface, as shown by the X-ray structure of the GFP dimer. Here, we show that A of just these two mutations in both fluorescent domains of ECFP–linker–EYFP constructs results in a fourfold increase in the EYFP-to-ECFP emission ratio, which yields a 16fold change in emission ratio upon protease cleavage of the peptide linker (Figure 1). Additional biophysical evidence is provided, which shows that the mutations indeed result in A of an intramolecular complex.

Supramolecular control of enzyme activity through cucurbit[8]uril-mediated dimerization.

At the double: Cucurbit[8]uril-mediated protein dimerization enables reversible control over strong enzyme activation of caspases. Simple addition of a short N-terminal FGG motif allows for a supramolecular-mediated 50-fold enhancement of caspase-9 catalytic activity.

High-Affinity Peptide-Based Collagen Targeting Using Synthetic Phage Mimics: From Phage Display to Dendrimer Display

Peptides derived from phage display typically show significantly weaker binding than their respective high affinity phage, which can bind to protein surfaces in a multivalent fashion. Here we show that mimicking key aspects of the multivalent architecture of the phage on an AB(5) dendritic wedge can enhance the affinity of a phage-display derived collagen binding peptide 100-fold (K(d) = 550 nM), allowing direct visualization of collagen architectures in native tissues with a higher specificity than that of the native collagen binding protein CNA35. The dendrimer display approach introduced here represents a well-defined, highly versatile platform for the affinity enhancement of phage display-derived peptides that is likely to be broadly applicable.

Imaging Collagen in Intact Viable Healthy and Atherosclerotic Arteries Using Fluorescently Labeled CNA35 and Two-Photon Laser Scanning Microscopy

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E−/− mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E−/− mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.

Fluorescent imaging of transition metal homeostasis using genetically encoded sensors.

The ability to image the concentration of transition metals in living cells in real time is important for understanding transition metal (TM) homeostasis and its involvement in diseases. Genetically encoded fluorescent sensor proteins are attractive because they do not require cell-invasive procedures, can be targeted to different locations in the cell, and allow ratiometric detection. Important progress in the development of Zn(2+) sensors has allowed sensitive detection of the very low free concentrations of Zn(2+) in single cells, both in the cytosol and various organelles. Together with other recent advances in chemical biology, these tools seem particularly useful to interrogate the dynamics and compartmentation of TM homeostasis.

Mitochondrial and ER-Targeted eCALWY Probes Reveal High Levels of Free Zn2+

Zinc (Zn2+) ions are increasingly recognized as playing an important role in cellular physiology. Whereas the free Zn2+ concentration in the cytosol has been established to be 0.1-1 nM, the free Zn2+ concentration in subcellular organelles is not well-established. Here, we extend the eCALWY family of genetically encoded Förster Resonance Energy Transfer (FRET) Zn2+ probes to permit measurements in the endo(sarco)plasmic reticulum (ER) and mitochondrial matrix. Deployed in a variety of mammalian cell types, these probes reveal resting mitochondrial free [Zn2+] values of ∼300 pM, somewhat lower than in the cytosol but 3 orders of magnitude higher than recently reported using an alternative FRET-based sensor. By contrast, free ER [Zn2+] was found to be ≥5 nM, which is >5000-fold higher than recently reported but consistent with the proposed role of the ER as a mobilizable Zn2+ store. Treatment of β-cells or cardiomyocytes with sarco(endo)plasmic reticulum Ca2+-ATPase inhibitors, mobilization of ER Ca2+ after purinergic stimulation with ATP, or manipulation of ER redox, exerted no detectable effects on [Zn2+]ER. These findings question the previously proposed role of Ca2+ in Zn2+ mobilization from the ER and suggest that high ER Zn2+ levels may be an important aspect of cellular homeostasis.