Franziska Zosel

Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments.

Significance In the interior of a cell, the volume accessible to each protein molecule is restricted by the presence of the large number of other macromolecules. Such a crowded environment is known to affect the stability and folding rates of proteins. In the case of intrinsically disordered proteins (IDPs), however, a class of proteins that lack stable structure, much less is known about the role of crowding effects. We have quantified the conformational changes occurring in IDPs in the presence of high concentrations of different polymers that act as crowding agents. Using single-molecule spectroscopy, we have identified effects that are typical of polymer solutions and have direct implications for the behavior of IDPs within the cell. Intrinsically disordered proteins (IDPs) are involved in a wide range of regulatory processes in the cell. Owing to their flexibility, their conformations are expected to be particularly sensitive to the crowded cellular environment. Here we use single-molecule Förster resonance energy transfer to quantify the effect of crowding as mimicked by commonly used biocompatible polymers. We observe a compaction of IDPs not only with increasing concentration, but also with increasing size of the crowding agents, at variance with the predictions from scaled-particle theory, the prevalent paradigm in the field. However, the observed behavior can be explained quantitatively if the polymeric nature of both the IDPs and the crowding molecules is taken into account explicitly. Our results suggest that excluded volume interactions between overlapping biopolymers and the resulting criticality of the system can be essential contributions to the physics governing the crowded cellular milieu.

Gas-Phase FRET Efficiency Measurements To Probe the Conformation of Mass-Selected Proteins

Electrospray ionization and mass spectrometry have revolutionized the chemical analysis of biological molecules, including proteins. However, the correspondence between a proteins native structure and its structure in the mass spectrometer (where it is gaseous) remains unclear. Here, we show that fluorescence (Förster) resonance energy transfer (FRET) measurements combined with mass spectrometry provides intramolecular distance constraints in gaseous, ionized proteins. Using an experimental setup which combines trapping mass spectrometry and laser-induced fluorescence spectroscopy, the structure of a fluorescently labeled mutant variant of the protein GB1 was probed as a function of charge state. Steady-state fluorescence emission spectra and time-resolved donor fluorescence measurements of mass-selected GB1 show a marked decrease in the FRET efficiency with increasing number of charges on the gaseous protein, which suggests a Coulombically driven unfolding and expansion of its structure. This lies in stark contrast to the pH stability of GB1 in solution. Comparison with solution-phase single-molecule FRET measurements show lower FRET efficiency for all charge states of the gaseous protein examined, indicating that the ensemble of conformations present in the gas phase is, on average, more expanded than the native form. These results represent the first FRET measurements on a mass-selected protein and illustrate the utility of FRET for obtaining a new kind of structural information for large, desolvated biomolecules.

Intramolecular Distances and Dynamics from the Combined Photon Statistics of Single-Molecule FRET and Photoinduced Electron Transfer

Single-molecule Förster resonance energy transfer (FRET) and photoinduced electron transfer (PET) have developed into versatile and complementary methods for probing distances and dynamics in biomolecules. Here we show that the two methods can be combined in one molecule to obtain both accurate distance information and the kinetics of intramolecular contact formation. In a first step, we show that the fluorescent dyes Alexa 488 and Alexa 594, which are frequently used as a donor and acceptor for single-molecule FRET, are also suitable as PET probes with tryptophan as a fluorescence quencher. We then performed combined FRET/PET experiments with FRET donor- and acceptor-labeled polyproline peptides. The placement of a tryptophan residue into the polyglycylserine tail incorporated in the peptides allowed us to measure both FRET efficiencies and the nanosecond dynamics of contact formation between one of the fluorescent dyes and the quencher. Variation of the linker length between the polyproline and the Alexa dyes and in the position of the tryptophan residue demonstrates the sensitivity of this approach. Modeling of the combined photon statistics underlying the combined FRET and PET process enables the accurate analysis of both the resulting transfer efficiency histograms and the nanosecond fluorescence correlation functions. This approach opens up new possibilities for investigating single biomolecules with high spatial and temporal resolution.

Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools

Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.

Single-molecule electrometry

Mass and electrical charge are fundamental properties of biological macromolecules. Although molecular mass has long been determined with atomic precision, a direct and precise determination of molecular charge remains an outstanding challenge. Here we report high-precision (<1e) measurements of the electrical charge of molecules such as nucleic acids, and globular and disordered proteins in solution. The measurement is based on parallel external field-free trapping of single macromolecules, permits the estimation of a dielectric coefficient of the molecular interior and can be performed in real time. Further, we demonstrate the direct detection of single amino acid substitution and chemical modifications in proteins. As the electrical charge of a macromolecule strongly depends on its three-dimensional conformation, this kind of high-precision electrometry offers an approach to probe the structure, fluctuations and interactions of a single molecule in solution.

The Most Stable Protein–Ligand Complex: Applications for One-Step Affinity Purification and Identification of Protein Assemblies†

The purification of protein complexes and large-scale investigations of protein–protein interaction networks have been greatly facilitated through the development of a number of affinity tags such as the cmyc, FLAG, and His6 tags. [2] However, all of the currently available affinity purification systems suffer from dynamic binding equilibria and measurable dissociation rate constants which enable the competitive elution of bound target proteins when an excess of a suitable free ligand is present. This circumstance hampers the quantitative and cost-effective isolation of low-abundance protein complexes. Here, we introduce a new affinity purification system that is derived from type 1 pili of E. coli. Type 1 pili are rigid, filamentous supramolecular protein complexes which are anchored in the cell s outer membrane and extend into the extracellular space. They are composed of four structural protein subunits termed FimH, FimG, FimF, and FimA. In the assembled pilus, these subunits interact noncovalently by a mechanism called donor strand complementation, where the incomplete, immunoglobulin-like fold of one subunit is completed by an N-terminal extension, termed donor strand, of the successive subunit. The complex between FimGt, an N-terminally truncated variant of FimG lacking its own donor strand, and a peptide corresponding to the donor strand of the neighboring subunit FimF (DsF) was found to be the kinetically most stable protein–ligand complex known to date (Figure 1). Here, we establish the FimGt/DsF system for use in the affinity purification of heterooligomeric protein complexes from cell extracts. Utilizing the donor strand of FimF as the affinity tag (termed DsF tag) and FimGt as the binding partner, we demonstrate the one-step purification of DsFtagged E. coli tryptophan synthase, a heterotetrameric abba complex of low cellular abundance. We compare the performance of the DsF tag to that of other commonly used affinity tags and find that, in agreement with the high kinetic stability of the FimGt/DsF complex, enrichment of the tryptophan synthase complex is most efficient for the DsF tag. This result suggests that the DsF tag is most suitable not only for the isolation of low-abundance protein complexes but presumably also for many other technical applications such as, for example, the functional and permanent immobilization of DsF-tagged proteins on surfaces and their detection in cells and on Western blot membranes. As a prerequisite for the technical application of the FimGt/DsF system we first optimized the production of FimGt by producing it in the cytoplasm of E. coli BL21(DE3) cells in the form of inclusion bodies (Figure S1 in the Supporting Information). After solubilization of the inclusion bodies, oxidative refolding, and purification of FimGt by conventional chromatographic techniques, the final yield of purified FimGt was 35 mg per liter of bacterial culture— sufficient amounts for large-scale applications of the FimGt/ DsF system. Quantitative formation of the single, structural disulfide bond was verified by the Ellman assay. The identity of FimGt was confirmed by ESI-MS (expected/measured mass: 13656.9/13657.0 Da). To gain mechanistic insight into the binding reaction between DsF and FimGt, association kinetics were measured for DsF concentrations of 5, 10, 25 and 50 mm while the FimGt concentration was kept constant at 5 mm (Figure 2 a). The reaction rates were dependent on the DsF concentration, indicating that binding of DsF is the rate-limiting step of complex formation. The data were globally fit according to an irreversible second-order reaction and yielded a rate constant of association of (330 8.9)m 1 s . Although relatively slow, the binding of DsF to FimGt is fast enough to allow for technical applications on reasonable time scales. We determined the rate constant for spontaneous dissociation/unfolding of the FimGt/DsF complex at pH 8.0 and 25 8C to be Figure 1. Crystal structure of the FimGt/DsF complex (3BFQ.pdb). FimGt is shown as a gray surface, the DsF peptide as stick representation. Residues of DsF that point towards FimGt are in bold and their position in the structure is indicated by arrows.

Rapid Microfluidic Double-Jump Mixing Device for Single-Molecule Spectroscopy

We introduce a microfluidic double-jump mixing device for investigating rapid biomolecular kinetics with confocal single-molecule spectroscopy. This device enables nonequilibrium dynamics to be probed, e.g., transiently populated intermediates that are inaccessible with existing single-molecule approaches. We demonstrate the potential and reliability of the method on time scales from milliseconds to minutes by investigating the coupled folding and binding reaction of two intrinsically disordered proteins and the conformational changes occurring in a large cytolytic pore-forming toxin.

Internal friction in an intrinsically disordered protein—Comparing Rouse-like models with experiments

Internal friction is frequently found in protein dynamics. Its molecular origin however is difficult to conceptualize. Even unfolded and intrinsically disordered polypeptide chains exhibit signs of internal friction despite their enormous solvent accessibility. Here, we compare four polymer theories of internal friction with experimental results on the intrinsically disordered protein ACTR (activator of thyroid hormone receptor). Using nanosecond fluorescence correlation spectroscopy combined with single-molecule Förster resonance energy transfer (smFRET), we determine the time scales of the diffusive chain dynamics of ACTR at different solvent viscosities and varying degrees of compaction. Despite pronounced differences between the theories, we find that all models can capture the experimental viscosity-dependence of the chain relaxation time. In contrast, the observed slowdown upon chain collapse of ACTR is not captured by any of the theories and a mechanistic link between chain dimension and internal friction is still missing, implying that the current theories are incomplete. In addition, a discrepancy between early results on homopolymer solutions and recent single-molecule experiments on unfolded and disordered proteins suggests that internal friction is likely to be a composite phenomenon caused by a variety of processes.

Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets. Here, we combine photoactivated light microscopy with quantitative statistical analysis to visualize protein distribution in intact cells. In particular, we focus on the localization of HRas and its minimal anchoring domain, CAAX. We demonstrate the existence of a complex partitioning behavior, where small domains coexist with larger ones. The protein content in these domains varied from two molecules to tens of molecules. We found that 40% of CAAX and 60% of HRas were localized in domains. Subsequently, we were able to manipulate protein distributions by inducing coalescence of supposedly cholesterol-enriched domains. Clustering resulted in an increase of the localized fraction by 15%.

Combining short- and long-range fluorescence reporters with simulations to explore the intramolecular dynamics of an intrinsically disordered protein

Intrinsically disordered proteins (IDPs) are increasingly recognized as a class of molecules that can exert essential biological functions even in the absence of a well-defined three-dimensional structure. Understanding the conformational distributions and dynamics of these highly flexible proteins is thus essential for explaining the molecular mechanisms underlying their function. Single-molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET) is a powerful tool for probing intramolecular distances and the rapid long-range distance dynamics in IDPs. To complement the information from FRET, we combine it with photoinduced electron transfer (PET) quenching to monitor local loop-closure kinetics at the same time and in the same molecule. Here we employed this combination to investigate the intrinsically disordered N-terminal domain of HIV-1 integrase. The results show that both long-range dynamics and loop closure kinetics on the sub-microsecond time scale can be obtained reliably from a single set of measurements by the analysis with a comprehensive model of the underlying photon statistics including both FRET and PET. A more detailed molecular interpretation of the results is enabled by direct comparison with a recent extensive atomistic molecular dynamics simulation of integrase. The simulations are in good agreement with experiment and can explain the deviation from simple models of chain dynamics by the formation of persistent local secondary structure. The results illustrate the power of a close combination of single-molecule spectroscopy and simulations for advancing our understanding of the dynamics and detailed mechanisms in unfolded and intrinsically disordered proteins.