18 research outputs found
Exploiting reversible interactions: hydrogels and protein cross-linkers
A series of low molecular weight thermoreversible cystine hydrogelators were
synthesised via solid-phase chemistry. Novel hydrogels were found to gelate at
concentrations of <2 mM using microwave super-heating. Benzoyl cystine amide
derivative hydrogel, which could form at a concentration of 0.5 mM, equivalent to
0.022% w/w of gelator with respect to water (an incredible 111,000 molecules of
water gelated per single molecule of gelator), was applied to cell culture of cervical
cancer (HeLa) cells, which were found to distribute within the gel. Hydrogels were
produced on a microarray format using a novel strategy involving deposition of
hydrogel solutions by inkjet printing. The incorporation of fluorescent dye
(Rhodamine B) into hydrogels provided a novel means for studying hydrogel
morphology.Reversible boronate chemistry was implemented for the capture and release of
proteins and peptides onto a solid-support as part of a modified peptide enrichment
strategy. The strategy was proven following synthesis of hydroxamic acid and
catechol modified peptides and a study of their interaction with solid-supported
phenylboronic acid. NHS active ester affinity tags and cross-linkers were synthesised
and applied to a 3D proteomics cross-linking analysis pipe-line. The introduction of a
PEG unit led to a cross-linker with increased hydrophilicity and improved
observation of both inter and intra-protein cross-links by mass spectrometry
Dual-bioorthogonal catalysis by a palladium peptide complex
Artificial metalloenzymes (ArMs) enrich bioorthogonal
chemistry
with new-to-nature reactions while limiting metal deactivation and
toxicity. This enables biomedical applications such as activating
therapeutics in situ. However, while combination
therapies are becoming widespread anticancer treatments, dual catalysis
by ArMs has not yet been shown. We present a heptapeptidic ArM with
a novel peptide ligand carrying a methyl salicylate palladium complex.
We observed that the peptide scaffold reduces metal toxicity while
protecting the metal from deactivation by cellular components. Importantly,
the peptide also improves catalysis, suggesting involvement in the
catalytic reaction mechanism. Our work shows how a palladium-peptide
homogeneous catalyst can simultaneously mediate two types of chemistry
to synthesize anticancer drugs in human cells. Methyl salicylate palladium
LLEYLKR peptide (2-Pd) succeeded to simultaneously produce
paclitaxel by depropargylation, and linifanib by SuzukiâMiyaura
cross-coupling in cell culture, thereby achieving combination therapy
on non-small-cell lung cancer (NSCLC) A549 cells
Optimized fragmentation regime for diazirine photo-cross-linked peptides
Cross-linking/mass spectrometry has
evolved into a robust technology
that reveals structural insights into proteins and protein complexes.
We leverage a new tribrid instrument with improved fragmentation capacities
in a systematic comparison to identify which fragmentation method
would be best for the identification of cross-linked peptides. Specifically,
we explored three fragmentation methods and two combinations: collision-induced
dissociation (CID), beam-type CID (HCD), electron-transfer dissociation
(ETD), ETciD, and EThcD. Trypsin-digested, SDA-cross-linked human
serum albumin (HSA) served as a test sample, yielding over all methods
and in triplicate analysis in total 2602 matched PSMs and 1390 linked
residue pairs at 5% false discovery rate, as confirmed by the crystal
structure. HCD wins in number of matched peptide-spectrum-matches
(958 PSMs) and identified links (446). CID is most complementary,
increasing the number of identified links by 13% (58 links). HCD wins
together with EThcD in cross-link site calling precision, with approximately
62% of sites having adjacent backbone cleavages that unambiguously
locate the link in both peptides, without assuming any cross-linker
preference for amino acids. Overall quality of spectra, as judged
by sequence coverage of both peptides, is best for EThcD for the majority
of peptides. Sequence coverage might be of particular importance for
complex samples, for which we propose a data dependent decision tree,
else HCD is the method of choice. The mass spectrometric raw data
has been deposited in PRIDE (PXD003737)
Blind Evaluation of Hybrid Protein Structure Analysis Methods based on Cross-Linking
Hybrid methods combine experimental data and computational modeling to analyze protein structures that are elusive to structure determination. To spur the development of hybrid methods, we propose to test them in the context of the CASP experiment and would like to invite experimental groups to participate in this initiative
Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics
Heterogeneous aggregates of the human protein α-synuclein (αSyn) are abundantly found in Lewy body inclusions of Parkinsonâs disease patients. While structural information on classical αSyn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of αSyn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-ÎČ amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict αSyn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining αSyn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative αSyn aggregation scavenges cytochrome câs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits
Complementary Benzophenone Cross-Linking/Mass Spectrometry Photochemistry
Use
of a heterobifunctional photoactivatable cross-linker, sulfo-SDA
(diazirine), has yielded high-density data that facilitated structure
modeling of individual proteins. We expand the photoactivatable chemistry
toolbox here with a second reagent, sulfo-SBP (benzophenone). This
further increases the density of photo-cross-linking to a factor of
20Ă over conventional cross-linking. Importantly, the two different
photoactivatable groups display orthogonal directionality, enabling
access to different protein regions, unreachable with a single cross-linker