Surface Attachment Enhances the Thermodynamic Stability of Protein L

Despite the importance of protein–surface interactions in both biology and biotechnology, our understanding of their origins is limited due to a paucity of experimental studies of the thermodynamics behind such interactions. In response, we have characterized the extent to which interaction with a chemically well‐defined macroscopic surface alters the stability of protein L. To do so, we site‐specifically attached a redox‐reporter‐modified protein variant to a hydroxy‐terminated monolayer on a gold surface and then used electrochemistry to monitor its guanidine denaturation and determine its folding free energy. Comparison with the free energy seen in solution indicates that interaction with this surface stabilizes the protein by 6 kJ mol^?1, a value that is in good agreement with theoretical estimates of the entropic consequences of surface‐induced excluded volume effects, thus suggesting that chemically specific interactions with this surface (e.g., electrostatic interactions) are limited in magnitude.     

Chiral Semiconductor Nanoparticles for Protein Catalysis and Profiling

In this study, via a simple one‐step method, chiral copper sulfide quantum dots (d/l‐QDs) were prepared using d‐/l‐penicillamine (d‐/l‐Pen). The anisotropy factor of d/l‐QDs was as high as 0.01. The d/l‐QDs can be used as photocatalysts to cleave proteins. Notably, the l‐QDs displayed the highest catalytic performance under left‐circularly polarized light irradiation. Mechanistic investigations indicate the generation of hydroxyl radicals as the reactive species that cause the cutting of proteins.     

Different Structural Origins of the Enantioselectivity of Haloalkane Dehalogenases toward Linear β‐Haloalkanes: Open–Solvated versus Occluded–Desolvated Active Sites

The enzymatic enantiodiscrimination of linear β‐haloalkanes is difficult because the simple structures of the substrates prevent directional interactions. Herein we describe two distinct molecular mechanisms for the enantiodiscrimination of the β‐haloalkane 2‐bromopentane by haloalkane dehalogenases. Highly enantioselective DbjA has an open, solvent‐accessible active site, whereas the engineered enzyme DhaA31 has an occluded and less solvated cavity but shows similar enantioselectivity. The enantioselectivity of DhaA31 arises from steric hindrance imposed by two specific substitutions rather than hydration as in DbjA.     

Ion Mobility Mass Spectrometry Measures the Conformational Landscape of p27 and its Domains and how this is Modulated upon Interaction with Cdk2/cyclin A

Intrinsically disordered proteins have been reported to undergo disorder‐to‐order transitions upon binding to their partners in the cell. The extent of the ordering upon binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclin A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behavior for function, particularly in the C‐terminal half of the protein. Herein, native ion mobility mass spectrometry (IM‐MS) is employed to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclin A. The trimeric Cdk2/cyclin A/p27‐KID complex possesses significant structural heterogeneity compared to Cdk2/cyclin A. These findings support the formation of a fuzzy complex in which both the N‐ and C‐termini of p27 interact with Cdk2/cyclin A in multiple, closely associated states.     

para‐Nitroblebbistatin,the Non‐Cytotoxic and Photostable Myosin II Inhibitor

Blebbistatin, the best characterized myosin II‐inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue‐light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity‐derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para‐nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.     

Chiral Amplification in Nature: Studying Cell‐Extracted Chiral Carotenoid Microcrystals via the Resonance Raman Optical Activity of Model Systems

Carotenoid microcrystals, extracted from cells of carrot roots and consisting of 95 % of achiral β‐carotene, exhibit a very intense chiroptical (ECD and ROA) signal. The preferential chirality of crystalline aggregates that consist mostly of achiral building blocks is a newly observed phenomenon in nature, and may be related to asymmetric information transfer from the chiral seeds (small amount of α‐carotene or lutein) present in carrot cells. To confirm this hypothesis, we synthesized several model aggregates from various achiral and chiral carotenoids. Because of the sergeant‐and‐soldier behavior, a small number of chiral sergeants (α‐carotene or astaxanthin) force the achiral soldier molecules (β‐ or 11,11′‐[D₂]‐β‐carotene) to jointly form supramolecular assemblies of induced chirality. The chiral amplification observed in these model systems confirmed that chiral microcrystals appearing in nature might consist predominantly of achiral building blocks and their supramolecular chirality might result from the co‐crystallization of chiral and achiral analogues.     

A Single Spherical Assembly of Protein Amyloid Fibrils Formed by Laser Trapping

Protein amyloids have received much attention owing to their correlation with serious diseases and to their promising mechanical and optical properties as future materials. Amyloid formation has been conducted by tuning temperature and chemical conditions, so that its nucleation and the following growth are analyzed as ensemble dynamics. A single spherical assembly of amyloid fibrils of cytochrome c domain‐swapped dimer was successfully generated upon laser trapping. The amyloid fibrillar structure was confirmed by fluorescence characterization and electron microscopy. The prepared spheres were further manipulated individually in solution to fabricate a three‐dimensional microstructure and a line pattern. Amyloid formation dynamics and amyloid‐based microstructure fabrication are demonstrated based on direct observation of a single spherical assembly, which foresees a new approach in amyloid studies.     

NMR Analysis of Apo Glutamine‐Binding Protein Exposes Challenges in the Study of Interdomain Dynamics

Glutamine‐binding protein (GlnBP) displays an apo, “open” and a holo, “closed” crystal form, mutually related by a rigid‐body reorientation of its domains. A fundamental question about such large‐scale conformational transitions, whether the closed state exists in the absence of ligand, is controversial in the case of GlnBP. NMR observations have indicated no evidence of the closed form, whereas experimentally validated computations have suggested a remarkable ca. 40 % population. Herein, a paramagnetic NMR strategy designed to detect the putative apo‐closed species shows that a major population of the latter is highly improbable. Further, NMR residual dipolar couplings collected under three anisotropic conditions do not reveal differential domain alignment and establish that the average solution conformation is satisfied by the apo‐open crystal structure. Our results indicate that the computational prediction of large‐scale interdomain motions is not trivial and may lead to erroneous conclusions without proper experimental validation.     

Substitution of the Catalytic Metal and Protein PEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase

Phosphotriesterase, a pesticide-degrading enzyme, from Flavobacterium sp. was cloned and expressed in Escherichia coli. The catalytic zinc ions were replaced by cobalt atoms increasing the catalytic activity of phosphotriesterase on different pesticides. This metal substitution increased the catalytic activity from 1.4 times to 4 times according to the pesticide. In order to explain this catalytic increase, QM/MM calculations were performed. Accordingly, the HOMO energy of the substrate is closer to the LUMO energy of the cobalt-substituted enzyme. The chemical modification of the enzyme surface with poly(ethylene glycol) increased the thermostability and stability against metal chelating agents of both metal phosphotriesterase preparations.     

Enantioselective Control of Both Helical and Axial Stereogenic Elements though an Organocatalytic Approach

A highly diastereo‐ and enantio‐selective method for the asymmetric synthesis of molecules containing helicenes and stereogenic axes was developed based on organocatalysis. Various compounds bearing both helical and axial stereogenic elements were obtained in excellent enantioselectivities. The mechanism study revealed that the reaction proceeded through two stages: 1) The first cyclization produces a reaction intermediate containing a stereogenic axis. 2) The dynamic kinetic resolution of helix reaction intermediate following with cyclization generates a helix and another stereogenic axis.     

Polyethylene Glycol Backfilling Mitigates the Negative Impact of the Protein Corona on Nanoparticle Cell Targeting

In protein‐rich environments such as the blood, the formation of a protein corona on receptor‐targeting nanoparticles prevents target recognition. As a result, the ability of targeted nanoparticles to selectively bind to diseased cells is drastically inhibited. Backfilling the surface of a targeted nanoparticle with polyethylene glycol (PEG) molecules is demonstrated to reduce the formation of the protein corona and re‐establishes specific binding. The length of the backfilled PEG molecules must be less than the length of the ligand linker; otherwise, PEG interferes with the binding of the targeting ligand to its corresponding cellular receptor.     

Gold Nanowire Chiral Ultrathin Films with Ultrastrong and Broadband Optical Activity

An ultrastrong and broadband chiroptical response is key but remains challenging for many device applications. A simple and cost‐effective bottom‐up method is introduced to fabricate large‐area long‐range ordered chiral ultrathin films with the Langmuir–Schaeffer technique using gold nanowires as building blocks. Significantly, as‐prepared ultrathin films display giant optical activity across a broad wavelength range covering visible and near infrared regions with an anisotropic factor of up to 0.285, which is the record value for bottom‐up techniques. Detailed experimental result and theoretical analysis disclose that such remarkable optical activity originates from birefringence and dichroism of the well‐aligned Au nanowire layers in the ultrathin films. The universality of this facile strategy for constructing chiral ultrathin films is further demonstrated with many other one‐dimensional nanomaterials.