Time-Resolved Ion Mobility Mass Spectrometry to Solve Conformational Changes in a Cryptochrome.

Many biological mechanisms rely on the precise control of conformational changes in proteins. Understanding such dynamic processes requires methods for determining structures and their temporal evolution. In this study, we introduce a novel approach to time-resolved ion mobility mass spectrometry. We validated the method on a simple photoreceptor model and applied it to a more complex system, the animal-like cryptochrome from Chlamydomonas reinhardtii (CraCRY), to determine the role of specific amino acids affecting the conformational dynamics as reaction to blue light activation. In our setup, using a high-power LED mounted in the source region of an ion mobility mass spectrometer, we allow a time-resolved evaluation of mass and ion mobility spectra. Cryptochromes like CraCRY are a widespread type of blue light photoreceptors and mediate various light-triggered biological functions upon excitation of their inbuilt flavin chromophore. Another hallmark of cryptochromes is their flexible carboxy-terminal extension (CTE), whose structure and function as well as the details of its interaction with the photolyase homology region are not yet fully understood and differ among different cryptochromes types. Here, we addressed the highly conserved C-terminal domain of CraCRY, to study the effects of single mutations on the structural transition of the C-terminal helix α22 and the attached CTE upon lit-state formation. We show that D321, the putative proton acceptor of the terminal proton-coupled electron transfer event from Y373, is essential for triggering the large-scale conformational changes of helix α22 and the CTE in the lit state, while D323 influences the timing.

Exploring the 2D-IR repertoire of the -SCN label to study site-resolved dynamics and solvation in the calcium sensor protein calmodulin.

The calcium sensor protein calmodulin is ubiquitous among eukaryotes. It translates intracellular Ca2+ influx (by a decrease of conformational flexibility) into increased target recognition affinity. Here we demonstrate that by using the IR reporter -SCN in combination with 2D-IR spectroscopy, global structure changes and local dynamics, degree of solvent exposure and protein-ligand interaction can be characterised in great detail. The long vibrational lifetime of the -SCN label allows for centerline slope analysis of the 2D-IR line shape up to 120 ps to deduce the frequency-frequency correlation function (FFCF) of the -SCN label in various states and label positions in the protein. Based on that we show clear differences between a solvent exposed site, the environment close to the Ca2+ binding motif and three highly conserved positions for ligand binding. Furthermore, we demonstrate how these dynamics are affected by conformational change induced by the addition of Ca2+ ions and by interaction with a short helical peptide mimicking protein binding. We show that the binding mode is strongly heterogeneous among the probed key binding methionine residues. SCN's vibrational relaxation is dominated by intermolecular contributions. Changes in the vibrational lifetime upon changing between H2O and D2O buffer therefore provide a robust measure for water accessibility of the label. Characterising -SCN's extinction coefficient, vibrational lifetime in light and heavy water and its FFCF we demonstrate the vast potential it has as a label especially for nonlinear spectroscopies, such as 2D-IR spectroscopy.

Local dynamics of the photo-switchable protein PYP in ground and signalling state probed by 2D-IR spectroscopy of -SCN labels.

Incorporation of minimally perturbative vibrational probes into proteins allows combination of the femtosecond time resolution of two dimensional infrared (2D-IR) spectroscopy with a spatial resolution on the level of single side chains. Here, we apply the thiocyanate (-SCN) label introduced by the cyanylation of cysteine to probe local dynamics in the photo-switchable protein PYP. We incorporated the -SCN label into five positions of the protein structure including PYP's core region, its solvent exposed surface and the chromophore-binding pocket. The analysis of -SCN's time dependent 2D-IR lineshape provides insight into the timescales and amplitudes of the dynamics in the label's protein and solvent microenvironment. We present a detailed analysis of the local protein dynamics found at all five labelling positions in PYP's dark state (pG). Absorption of a blue photon triggers the isomerisation of PYP's chromophore and eventually leads to an overall reorganisation of the protein structure, where PYP ends up in a less structured signalling state pB. Employing 2D-IR spectroscopy also on the signalling state allows assessment of the change of local dynamics compared to the pG state.

Peptidomimetics That Inhibit and Partially Reverse the Aggregation of Aß1-42.

The peptide sequence KLVFF resembles the hydrophobic core of the Aß peptide known to form amyloid plaques in Alzheimer's disease. Starting from its retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step by step natural amino acids have been replaced by aromatic building blocks accessible from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against proteolytic decay and largely prevents the aggregation of Aß1-42 over extended periods of time. The activity of the new inhibitors was tested first by fluorescence correlation spectroscopy. For closer examination of compound 18, additional techniques were also applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aß but also can partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.

Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation.

Cysteine conjugation is an important tool in protein research and relies on fast, mild and chemoselective reactions. Cysteinyl thiols can either be modified with prefunctionalized electrophiles, or converted into electrophiles themselves for functionalization with selected nucleophiles in an independent step. Here we report a bioconjugation strategy that uses a vinyl thianthrenium salt to transform cysteine into a highly reactive electrophilic episulfonium intermediate in situ, to enable conjugation with a diverse set of bioorthogonal nucleophiles in a single step. The reactivity profile can connect several nucleophiles to biomolecules through a short and stable ethylene linker, ideal for introduction of infrared labels, post-translational modifications or NMR probes. In the absence of reactive exogenous nucleophiles, nucleophilic amino acids can react with the episulfonium intermediate for native peptide stapling and protein-protein ligation. Ready synthetic access to isotopologues of vinyl thianthrenium salts enables applications in quantitative proteomics. Such diverse applications demonstrate the utility of vinyl-thianthrenium-based bioconjugation as a fast, selective and broadly applicable tool for chemical biology.

Bacterial F-type ATP synthases follow a well-choreographed assembly pathway.

F-type ATP synthases are multiprotein complexes composed of two separate coupled motors (F1 and FO) generating adenosine triphosphate (ATP) as the universal major energy source in a variety of relevant biological processes in mitochondria, bacteria and chloroplasts. While the structure of many ATPases is solved today, the precise assembly pathway of F1FO-ATP synthases is still largely unclear. Here, we probe the assembly of the F1 complex from Acetobacterium woodii. Using laser induced liquid bead ion desorption (LILBID) mass spectrometry, we study the self-assembly of purified F1 subunits in different environments under non-denaturing conditions. We report assembly requirements and identify important assembly intermediates in vitro and in cellula. Our data provide evidence that nucleotide binding is crucial for in vitro F1 assembly, whereas ATP hydrolysis appears to be less critical. We correlate our results with activity measurements and propose a model for the assembly pathway of a functional F1 complex.

Cotranslational assembly of membrane protein/nanoparticles in cell-free systems.

Nanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins. We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.

Structural rearrangement of amyloid-ß upon inhibitor binding suppresses formation of Alzheimer's disease related oligomers.

The formation of oligomers of the amyloid-ß peptide plays a key role in the onset of Alzheimer's disease. We describe herein the investigation of disease-relevant small amyloid-ß oligomers by mass spectrometry and ion mobility spectrometry, revealing functionally relevant structural attributes. In particular, we can show that amyloid-ß oligomers develop in two distinct arrangements leading to either neurotoxic oligomers and fibrils or non-toxic amorphous aggregates. Comprehending the key-attributes responsible for those pathways on a molecular level is a pre-requisite to specifically target the peptide's tertiary structure with the aim to promote the emergence of non-toxic aggregates. Here, we show for two fibril inhibiting ligands, an ionic molecular tweezer and a hydrophobic peptide that despite their different interaction mechanisms, the suppression of the fibril pathway can be deduced from the disappearance of the corresponding structure of the first amyloid-ß oligomers.