15 research outputs found
Counting tagged molecules one by one: Quantitative photoactivation and bleaching of photoactivatable fluorophores
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Synthesis and application of the blue fluorescent amino acid l -4-cyanotryptophan to assess peptideâmembrane interactions
Recently, l-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected l-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide-membrane binding constants
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Glutamine Side Chain 13Câ18O as a Nonperturbative IR Probe of Amyloid Fibril Hydration and Assembly.
Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain 13Câ18O, have been widely employed to obtain site-specific structural information, yet only secondary structures and strand-to-strand arrangements can be probed. Very few nonperturbative IR probes are available to report on the side-chain conformation and environments, which are critical to determining sheet-to-sheet arrangements in steric zippers within amyloids. Polar residues, such as glutamine, contribute significantly to the stability of amyloids and thus are frequently found in core regions of amyloid peptides/proteins. Furthermore, polyglutamine (polyQ) repeats form toxic aggregates in several neurodegenerative diseases. Here we report the synthesis and application of a new nonperturbative IR probe-glutamine side chain 13Câ18O. We use side chain 13Câ18O labeling and isotope dilution to detect the presence of intermolecularly hydrogen-bonded arrays of glutamine side chains (Gln ladders) in amyloid-forming peptides. Moreover, the line width of the 13Câ18O peak is highly sensitive to its local hydration environment. The IR data from side chain labeling allows us to unambiguously determine the sheet-to-sheet arrangement in a short amyloid-forming peptide, GNNQQNY, providing insight that was otherwise inaccessible through main chain labeling. With several different fibril samples, we also show the versatility of this IR probe in studying the structures and aggregation kinetics of amyloids. Finally, we demonstrate the capability of modeling amyloid structures with IR data using the integrative modeling platform (IMP) and the potential of integrating IR with other biophysical methods for more accurate structural modeling. Together, we believe that side chain 13Câ18O will complement main chain isotope labeling in future IR studies of amyloids and integrative modeling using IR data will significantly expand the power of IR spectroscopy to elucidate amyloid assemblies
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Designed peptides that assemble into cross-α amyloid-like structures
Designed peptides that assemble into cross-alpha amyloid-like structures
Amyloids adopt `cross-beta' structures composed of long, twisted fibrils
with beta-strands running perpendicular to the fibril axis. Recently, a
toxic peptide was proposed to form amyloid-like cross-alpha structures
in solution, with a planar bilayer-like assembly observed in the crystal
structure. Here we crystallographically characterize designed peptides
that assemble into spiraling cross-alpha amyloid-like structures, which
resemble twisted beta-amyloid fibrils. The peptides form helical dimers,
stabilized by packing of small and apolar residues, and the dimers
further assemble into cross-alpha amyloid-like fibrils with superhelical
pitches ranging from 170 angstrom to 200 angstrom. When a small residue
that appeared critical for packing was converted to leucine, it resulted
in structural rearrangement to a helical polymer. Fluorescently tagged
versions of the designed peptides form puncta in mammalian cells, which
recover from photobleaching with markedly different kinetics. These
structural folds could be potentially useful for directing in vivo
protein assemblies with predetermined spacing and stabilities
Constructing ion channels from water-soluble α-helical barrels
The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptideâlipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide
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Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors