Search

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ

Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain (13)C=(18)O, 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 (13)C=(18)O. We use side chain (13)C=(18)O 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 (13)C=(18)O 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 (13)C=(18)O 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.

Water is essential for protein folding and assembly of amyloid fibrils. Internal water cavities have been proposed for several amyloid fibrils, but no direct structural and dynamical data have been reported on the water dynamics and site-specific interactions of water with the fibrils. Here we use solid-state NMR spectroscopy to investigate the water interactions of several Aβ40 fibrils. 1H spectral lineshapes, T2 relaxation times, and two-dimensional (2D) 1H–13C correlation spectra show that there are five distinct water pools: three are peptide-bound water, while two are highly dynamic water that can be assigned to interfibrillar water and bulk-like matrix water. All these water pools are associated with the fibrils on the nanometer scale. Water-transferred 2D correlation spectra allow us to map out residue-specific hydration and give evidence for the presence of a water pore in the center of the three-fold symmetric wild-type Aβ40 fibril. In comparison, the loop residues and the intramolecular strand–strand interface have low hydration, excluding the presence of significant water cavities in these regions. The Osaka Aβ40 mutant shows lower hydration and more immobilized water than wild-type Aβ40, indicating the influence of peptide structure on the dynamics and distribution of hydration water. Finally, the highly mobile interfibrillar and matrix water exchange with each other on the time scale of seconds, suggesting that fibril bundling separates these two water pools, and water molecules must diffuse along the fibril axis before exchanging between these two environments. These results provide insights and experimental constraints on the spatial distribution and dynamics of water pools in these amyloid fibrils.

Inhibition of integrin alpha5beta1 emerges as a novel therapeutic option to block transmission of contractile forces during asthma attack. We designed and synthesized novel inhibitors of integrin alpha5beta1 by backbone replacement of known alphavbeta1 integrin inhibitors. These integrin alpha5beta1 inhibitors also retain the nanomolar potency against alphavbeta1 integrin, which shows promise for developing dual integrin alpha5beta1/alphavbeta1 inhibitor. Introduction of hydrophobic adamantane group significantly boosted the potency as well as selectivity over integrin alphavbeta3. We also demonstrated one of the inhibitors (11) reduced airway hyperresponsiveness in Ex vivo mouse tracheal ring assay. Results from this study will help guide further development of integrin alpha5beta1 inhibitors as potential novel asthma therapeutics.

The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

Although helices play key roles in peptide–protein and protein–protein interactions, the helical conformation is generally unstable for short peptides (10–15 residues) in aqueous solution in the absence of their binding partners. Thus, stabilizing the helical conformation of peptides can lead to increases in binding potency, specificity, and stability towards proteolytic degradation. Helices have been successfully stabilized by introducing side chain-to-side chain crosslinks within the central portion of the helix. However, this approach leaves the ends of the helix free, thus leading to fraying and exposure of the non-hydrogen-bonded amide groups to solvent. Here, we develop a “capped-strapped” peptide strategy to stabilize helices by embedding the entire length of the helix within a macrocycle, which also includes a semirigid organic template as well as end-capping interactions. We have designed a ten-residue capped-strapped helical peptide that behaves like a miniprotein, with a cooperative thermal unfolding transition and T(m) ≈70°C, unprecedented for helical peptides of this length. The NMR structure determination confirmed the design, and X-ray crystallography revealed a novel quaternary structure with implications for foldamer design.

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

Antibodies containing a fluorescent label or recombinant proteins containing a fluorescent reporter are commonly used to visualize proteins in situ. However, antibodies and bulky reporters can perturb protein structure and function. To overcome this problem, we have designed a minimally perturbing blue fluorescent unnatural amino acid that enables the direct imaging of intrinsically-fluorescent proteins with their interaction partners. Specifically, we used a nitrile-derivatized tryptophan, 4-cyanotryptophan (4CNTrp), which differs by only two atoms from native tryptophan. 4CNTrp has unique photophysical properties in the visible blue region: an absorption maximum at ~325 nm, an emission maximum at ~420 nm, a large fluorescence quantum yield (>0.8 in aqueous solution), a long fluorescence lifetime (ca. 13 ns), and good photostability (Hilaire et al. PNAS 2017; 114: 6005-09). Based on these properties, 4CNTrp-labeling was recently used to visualize the binding of a model peptide pHLIP (pH-(Low) Insertion Peptide) to cell membranes via wide-field fluorescence microscopy. 4CNTrp is also a viable FRET donor for acceptor pairs in the green visible region. 4CNTrp can be prepared by a simple, high-yielding cost-effective synthetic route and because it does not inhibit bacterial growth, it may be practical for cellular applications (Zhang et al. Chem. Comm. 2019; 55: 5095-98). Here, we used 4CNTrp-labeled peptides to directly image integrins on cell surfaces. Previously, we described the synthesis of peptides, called CHAMP peptides for Computed Helical Anti-Membrane Protein, that target the transmembrane (TM) domains of integrins in a sequence-specific manner (Yin et al. Science 2007; 315: 1817-22). Thus, the CHAMP peptides anti-αIIb and anti-αv specifically bind to the TM domains of αIIb and αv in platelet membranes, causing separation of the αIIbβ3 and αvβ3 TM domains and αIIbβ3 and αvβ3 activation. Anti-αIIb also caused αIIbβ3 clustering that was visualized using the β3-specific monoclonal antibody SSA6 labeled with Alexa Fluor 488 but only after the platelets were fixed with paraformaldehyde to prevent antibody-induced clustering. To enable the direct visualization of αIIβ3, αvβ3, and α2β1 on cell surfaces, we used solid-phase peptide synthesis to generate CHAMP peptides labeled with 4CNTrp at their N-termini (CN-αIIb, CN-αv, CN-β1). We found that 4CNTrp labeling did not perturb the ability of the CHAMP peptides to bind to integrins and to cause specific integrin activation. Thus, CN-αIIb caused αIIbβ3-dependent platelet aggregation, CN-αv caused the αvβ3-dependent platelet adhesion to osteopontin, and CN-α1 caused α2β1-mediated platelet adhesion to collagen. We then used high resolution wide-field deconvolution microscopy to image 4CNTrp-containing integrins on the surface of platelets, HEL cells, and transfected CHO cells. Compared to 4CNTrp-containing random peptides that uniformly decorate cell surfaces, the CN-labeled CHAMP peptides were present as a limited number of discrete foci on the cell surface. To confirm that these foci represented CN-peptide containing integrins, we co-stained the cells with Alexa Fluor 488-labeled integrin-specific monoclonal antibodies and found that CN-peptide and antibody fluorescence coincided. Thus, these studies demonstrate the specific and direct imaging of integrins embedded in cell membranes in their activated state using 4CNTrp-labeled anti-integrin TM peptides. Because 4CNTrp can readily be incorporated into proteins and peptides with little if any structural perturbation, 4CNTrp-labeling provides a facile way to directly monitor protein behavior and protein-protein interactions in cellular environments. Disclosures No relevant conflicts of interest to declare.

One small molecule inhibitor of αvβ1 integrin, c8, shows antifibrotic effects in multiple in vivo mouse models. Here we synthesized c8 analogues and systematically investigate their structure-activity relationships (SAR) in αvβ1 integrin inhibition. N-Phenylsulfonyl-l-homoproline analogues of c8 maintained excellent potency against αvβ1 integrin while retaining good selectivity over other RGD integrins. In addition, 2-aminopyridine or cyclic guanidine analogues were shown to be equally potent to c8. A rigid phenyl linker increased the potency compared to c8, but the selectivity over other RGD integrins diminished. These results can provide further insights on design of αvβ1 integrin inhibitors as antifibrotics.

The amyloid-β (Aβ) peptide of Alzheimer's disease (AD) forms polymorphic fibrils on the micrometer and molecular scales. Various fibril growth conditions have been identified to cause polymorphism, but the intrinsic amino acid sequence basis for this polymorphism has been unclear. Several single-site mutations in the center of the Aβ sequence cause different disease phenotypes and fibrillization properties. The E22G (Arctic) mutant is found in familial AD and forms protofibrils more rapidly than wild-type Aβ. Here, we use solid-state NMR spectroscopy to investigate the structure, dynamics, hydration and morphology of Arctic E22G Aβ40 fibrils. (13)C, (15)N-labeled synthetic E22G Aβ40 peptides are studied and compared with wild-type and Osaka E22Δ Aβ40 fibrils. Under the same fibrillization conditions, Arctic Aβ40 exhibits a high degree of polymorphism, showing at least four sets of NMR chemical shifts for various residues, while the Osaka and wild-type Aβ40 fibrils show a single or a predominant set of chemical shifts. Thus, structural polymorphism is intrinsic to the Arctic E22G Aβ40 sequence. Chemical shifts and inter-residue contacts obtained from 2D correlation spectra indicate that one of the major Arctic conformers has surprisingly high structural similarity with wild-type Aβ42. (13)C-(1)H dipolar order parameters, (1)H rotating-frame spin-lattice relaxation times and water-to-protein spin diffusion experiments reveal substantial differences in the dynamics and hydration of Arctic, Osaka and wild-type Aβ40 fibrils. Together, these results strongly suggest that electrostatic interactions in the center of the Aβ peptide sequence play a crucial role in the three-dimensional fold of the fibrils, and by inference, fibril-induced neuronal toxicity and AD pathogenesis.

Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening twenty-four commercially available crosslinkers for successful cysteine alkylation in a model peptide sequence. The effects of crosslinking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid crosslinkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs and will be useful for future investigation of calpains, while the crosslinking technique will enable exploration of other protein-protein interactions.

Thioamides are sterically almost identical to their oxoamide counterparts, but are weaker hydrogen bond acceptors in comparison. Therefore, thioamide amino acids are excellent candidates for perturbing the energetics of backbone-backbone hydrogen bonds in proteins and hence should be useful in elucidating protein folding mechanisms in a site-specific manner. Herein, we validate this approach by applying it to probe the dynamic role of interstrand hydrogen bond formation in the folding kinetics of a well-studied β-hairpin, tryptophan zipper. Our results show that reducing the strength of the peptide’s backbone-backbone hydrogen bonds, except the one directly next to the β-turn, does not change the folding rate, suggesting that most native interstrand hydrogen bonds in β-hairpins are formed only after the folding transition state.

The nitrile stretching vibration is increasingly used as a sensitive infrared probe of local protein environments. However, site-specific incorporation of a nitrile moiety into proteins is difficult. Here we show that various aromatic nitriles can be easily incorporated into peptides and proteins via either thiol alkylation or arylation reaction.

Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.

Irradiation of the enone benzothiazoline 3 leads to the formation of cyclobutane 5. Preliminary mechanistic studies establish the intermediacy of an enecarbamate 14 in this photochemical transformation, which could be the result of sulfur extrusion from an episulfide intermediate. Photocycloaddition of the enecarbamate intermediate 14 leads to the formation of “crossed” photoadducts, i.e., 5, in excellent yield, with high levels of regio- and stereochemical control.

A highly stereoselective and efficient asymmetric total synthesis of (+)-laurencin (1) has been accomplished from the known oxazolidinone 5 in 15 steps. The route features an efficient internal alkylation to form oxocene 3 from 4 and a novel use of acetonitrile anion as a two-carbon acetaldehyde equivalent for direct synthesis of ketone 2 from alpha-alkoxy amide 3.

A concise and efficient synthesis of the trans-hydrindane system has been achieved by a novel intramolecular SN2′ enolate alkylation/ring-closing metathesis (RCM) strategy with a good degree of stereocontrol.