Nanoporous frameworks are polymeric materials built from rigid molecules, which give rise to their nanoporous structures with applications in gas sorption and storage, catalysis and others. ...Conceptually new applications could emerge, should these beneficial properties be manipulated by external stimuli in a reversible manner. One approach to render nanoporous frameworks responsive to external signals would be to immobilize molecular switches within their nanopores. Although the majority of molecular switches require conformational freedom to isomerize, and switching in the solid state is prohibited, the nanopores may provide enough room for the switches to efficiently isomerize. Here we describe two families of nanoporous materials incorporating the spiropyran molecular switch. These materials exhibit a variety of interesting properties, including reversible photochromism and acidochromism under solvent-free conditions, light-controlled capture and release of metal ions, as well reversible chromism induced by solvation/desolvation.
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► Homonuclear J-decoupling is achieved by exploiting naturally occurring 13Cs. ► Trains of BIRD pulses refocusing 1H(13C)–1H(12C) couplings leave only chemical shifts. ► Such ...experiment is refined to acquire “pure-shift” 1D 1H spectra. ► Considerations to implement the resulting experiment with robustness are described.
Achieving homonuclear 1H decoupling remains one of the key challenges in liquid-state NMR. Such spectra would endow a variety of organic and analytical applications with an increased resolution, and would ideally do so even in a one-dimensional format. A number of parallel efforts aimed at achieving this goal using two-dimensional acquisitions have been proposed; approaches demonstrated over recent years include, among others, new modes for achieving purely-absorptive J spectroscopy, the use of spatially-selective manipulations, and exploiting the natural spin dilution afforded by heteronuclei. The present study relies on the latter approach, and explores the use of BIRD pulses distinguishing between protons bonded to 13C from those bonded to 12C, to achieve homonuclear decoupling in a continuous 1D scan. Studies on several representative compounds demonstrate that this goal can be implemented in a robust format, provided that suitable care is also taken to suppress unwanted coherences, of making all manipulations sufficiently broad-banded, and to provide adequate heteronuclear decoupling of the targeted protons. Dependable homonuclear decoupling performance can then be achieved, with minimal line width, fine-tuning, and sensitivity penalties.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals ...of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW’s enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-¹H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis. Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting ¹H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a highaffinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino ¹H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.
This study describes the design and evaluation of a portable bright-field and fluorescence microscope that can be manufactured for $240 USD. The microscope uses a battery-operated LED-based ...flashlight as the light source and achieves a resolution of 0.8 microm at 1000x magnification in fluorescence mode. We tested the diagnostic capability of this new instrument to identify infections caused by the human pathogen, Mycobacterium tuberculosis. Sixty-four direct, decontaminated, and serially diluted smears were prepared from sputa obtained from 19 patients suspected to have M. tuberculosis infection. Slides were stained with auramine orange and evaluated as being positive or negative for M. tuberculosis with both the new portable fluorescence microscope and a laboratory grade fluorescence microscope. Concordant results were obtained in 98.4% of cases. This highly portable, low cost, fluorescence microscope may be a useful diagnostic tool to expand the availability of M. tuberculosis testing at the point-of-care in low resource settings.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
While
C-based Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) experiments offer an attractive alternative for establishing molecular structures, they suffer from low ...sensitivities arising from the scarcity of spin pairs present at natural abundance. Herein we demonstrate that dissolution dynamic nuclear polarization (dDNP) provides sufficient sensitivity to acquire 1D
C INADEQUATE spectra in a single scan and at natural abundance. Moreover, if steps are adopted to endow sub-Hertz precision to these measurements, they allow one to measure carbon-carbon J couplings over both one and multiple bonds for each chemical site. As these J
-couplings are usually sufficiently distinct to enable univocal pairing of the nuclei involved, essentially the same information as in 2D INADEQUATE can be obtained. The feasibility of the method is demonstrated for a range of compounds, including natural products such as α-pinene, menthol and limonene. Features and extensions of this approach are briefly discussed.
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IJS, KILJ, NUK, PNG, UL, UM
A method to detect NMR spectra from heteronuclei through the modulation that they impose on a water resonance is exemplified. The approach exploits chemical exchange saturation transfers, which can ...magnify the signal of labile protons through their influence on a water peak. To impose a heteronuclear modulation on water, an HMQC‐type sequence was combined with the FLEX approach. 1D 15N NMR spectra of exchanging sites could thus be detected, with about tenfold amplifications over the 15N modulations afforded by conventionally detected HMQC NMR spectroscopy. Extensions of this approach enable 2D heteronuclear acquisitions on directly bonded 1H–15N spin pairs, also with significant signal amplification. Despite the interesting limits of detection that these signal enhancements could open in NMR spectroscopy, these gains are constrained by the rates of solvent exchange of the targeted heteronuclear pairs, as well as by spectrometer instabilities affecting the intense water resonances detected in these experiments.
NMR resonances from heteronuclei can be detected through the modulation that they impose on a strong H2O resonance (shown in red) by combining a 2D heteronuclear NMR sequence with a 2D NMR approach relying on chemical exchange saturation transfer principles (HetFLEX). 1D 15N and 2D 1H/15N NMR spectra of small molecules and peptides can be recorded with substantial amplification.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Thanks to their special spatiotemporal encoding/decoding scheme, ultrafast (UF) NMR sequences can deliver arbitrary 2D spectra following a single excitation. Regardless of their nature, these ...sequences have in common their tracing of a path in the
F
1
–
t
2
plane, that will deliver the spectrum being sought after a 1D Fourier transformation versus
t
2
. This need to simultaneously digitize two domains, tends to impose bandwidth limitations along all spectral axes. Along the
t
2
/
F
2
dimension this problem is exacerbated by the fact that odd and even time points are not equispaced, and by additional artifacts such as time shifts between time points sampled while under the action of positive and negative decoding gradients. As a result, odd and even
t
2
points are typically Fourier transformed separately, halving the potential spectral width along this dimension. While this halving of the
F
2
span can be overcome by an interlaced Fourier transform, this post-processing is seldom used because of its sensitivity to hardware inaccuracies requiring even finer corrections of the even/odd
t
2
data points. These corrections have so far been done manually, but are challenging to implement when dealing with low signal-to-noise ratio signals like those associated with biomolecular NMR experiments. This study introduces an algorithm for an automatic correction of all even/odd ultrafast NMR inconsistencies, based on the acquisition of a reference scan on the solvent. This algorithm was verified experimentally using an
1
H
-
13
C
UF-HSQC variant on ubiquitin at 600 MHz. Features of this method as well as of the interlaced Fourier transformation in general, are discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target ...a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells. These molecules are highly ligand-efficient (MW < 330) and display specific biochemical and cellular activity by suppressing the maturation of miR-21, thereby providing an avenue toward therapeutic development in multiple diseases where miR-21 is abnormally expressed.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. ...Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.
Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In ...this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and
1
H-
15
N 2D correlation experiments. Here we introduce 2D
13
C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform ‘hyperpolarization-selective’ signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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