Gregory L. Olsen

Toward single-shot pure-shift solution 1H NMR by trains of BIRD-based homonuclear decoupling.

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 (13)C from those bonded to (12)C, 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.

Nanoporous frameworks exhibiting multiple stimuli responsiveness

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.

Monitoring Mechanistic Details in the Synthesis of Pyrimidines via Real-Time, Ultrafast Multidimensional NMR Spectroscopy

Recent years have witnessed unprecedented advances in the development of fast multidimensional NMR acquisition techniques. This progress could open valuable new opportunities for the elucidation of chemical and biochemical processes. This study demonstrates one such capability, with the first real-time Two-dimensional (2D) dynamic analysis of a complex organic reaction relying on unlabeled substrates. Implementing such measurements required the development of new ultrafast 2D methods, capable of monitoring multiple spectral regions of interest as the reaction progressed. The alternate application of these acquisitions in an interleaved, excitation-optimized fashion, allowed us to extract new structural and dynamic insight concerning the reaction between aliphatic ketones and triflic anhydride in the presence of nitriles to yield alkylpyrimidines. Up to 2500 2D NMR data sets were thus collected over the course of this nearly 100 min long reaction, in an approach resembling that used in functional magnetic resonance imaging. With the aid of these new frequency-selective low-gradient strength experiments, supplemented by chemical shift calculations of the spectral coordinates observed in the 2D heteronuclear correlations, previously postulated intermediates involved in the alkylpyrimidine formation process could be confirmed, and hitherto undetected ones were revealed. The potential and limitations of the resulting methods are discussed.

Theory of nonrigid rotational motion applied to NMR relaxation in RNA.

Solution NMR spectroscopy can elucidate many features of the structure and dynamics of macromolecules, yet relaxation measurements, the most common source of experimental information on dynamics, can sample only certain ranges of dynamic rates. A complete characterization of motion of a macromolecule thus requires the introduction of complementary experimental approaches. Solid-state NMR spectroscopy successfully probes the time scale of nanoseconds to microseconds, a dynamic window where solution NMR results have been deficient, and probes conditions where the averaging effects of rotational diffusion of the molecule are absent. Combining the results of the two distinct techniques within a single framework provides greater insight into dynamics, but this task requires the common interpretation of results recorded under very different experimental conditions. Herein, we provide a unified description of dynamics that is robust to the presence of large-scale conformational exchange, where the diffusion tensor of the molecule varies on a time scale comparable to rotational diffusion in solution. We apply this methodology to the HIV-1 TAR RNA molecule, where conformational rearrangements are both substantial and functionally important. The formalism described herein is of greater generality than earlier combined solid-state/solution NMR interpretations, if detailed molecular structures are available, and can offer a more complete description of RNA dynamics than either solution or solid-state NMR spectroscopy alone.

Slow exchange model of nonrigid rotational motion in RNA for combined solid-state and solution NMR studies.

Functional RNA molecules are conformationally dynamic and sample a multitude of dynamic modes over a wide range of frequencies. Thus, a comprehensive description of RNA dynamics requires the inclusion of a broad range of motions across multiple dynamic rates which must be derived from multiple spectroscopies. Here we describe a slow conformational exchange theoretical approach to combining the description of local motions in RNA that occur in the nanosecond to microsecond window and are detected by solid-state NMR with nonrigid rotational motion of the HIV-1 transactivation response element (TAR) RNA in solution as observed by solution NMR. This theoretical model unifies the experimental results generated by solution and solid-state NMR and provides a comprehensive view of the dynamics of HIV-1 TAR RNA, a well-known paradigm of an RNA where function requires extensive conformational rearrangements. This methodology provides a quantitative atomic level view of the amplitudes and rates of the local and collective displacements of the TAR RNA molecule and provides directly motional parameters for the conformational capture hypothesis of this classical RNA-ligand interaction.

Heteronuclear 1D and 2D NMR Resonances Detected by Chemical Exchange Saturation Transfer to Water

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 15 N NMR spectra of exchanging sites could thus be detected, with about tenfold amplifications over the 15 N modulations afforded by conventionally detected HMQC NMR spectroscopy. Extensions of this approach enable 2D heteronuclear acquisitions on directly bonded 1 H-15 N 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.

Correction to "Theory of nonrigid rotational motion applied to NMR relaxation in RNA".

In the following manuscript, we aim to provide corrections to expressions previously published in the Journal of Physical Chemistry A.1 This original manuscript (OM) sought to provide a bridge between solid-state NMR-derived motional models and solution experimental results by deriving an expression for the transition probability of the orientation of an atomic bond attached to a molecule that is undergoing both (a) overall rotational diffusion, and (b) an exchange between two or more different conformational states. The procedure involved finding an eigenfunction expansion of the transition probability for a single conformer, and then generalizing the time dependence of this expansion to incorporate an exchange between conformers with different rotational diffusion tensors (under the assumption of collinear diffusion tensors at the moment of exchange; for more on this, see the Discussion section). The proposed corrections apply to the latter step of finding the generalized time dependent coefficients. The OM expressions do not explicitly show the dependence of the chosen single conformer eigenfunctions on the diffusion tensor of the given conformer. An oversight on the part of the authors in this regard resulted in incorrect expressions for (a) the time evolution equation for these time-dependent coefficients (Eq. 18) and (b) the diffusion tensor dependences in the correlation function (Eq. 21). In the following sections, we remedy these equations and illustrate the results of utilizing the new expressions in our simulations of the solution relaxation times T1 and T2. It will be shown that differences between the new simulation results and those in the OM are much smaller than the experimental errors in the relaxation times, implying that the discussions of the physical implications of the results presented in the OM remain entirely valid. The first section provides a detailed re-derivation of the equations in question to clarify the points of difference between the OM and the new expressions. The next section presents the new simulation results, together with graphs of “residuals” between the OM relaxation times and the relaxation times calculated using the updated expressions.

High-Resolution 2D NMR of Disordered Proteins Enhanced by Hyperpolarized Water

This study demonstrates the usefulness derived from relying on hyperpolarized water obtained by dissolution DNP, for site-resolved biophysical NMR studies of intrinsically disordered proteins. Thanks to the facile amide-solvent exchange experienced by protons in these proteins, 2D NMR experiments that like HMQC rely on the polarization of the amide protons, can be enhanced using hyperpolarized water by several orders of magnitude over their conventional counterparts. Optimizations of the DNP procedure and of the subsequent injection into the protein sample are necessary to achieve these gains while preserving state-of-the-art resolution; procedures enabling this transfer of the hyperpolarized water and the achievement of foamless hyperpolarized protein solutions are demonstrated. These protocols are employed to collect 2D 15N-1H HMQC NMR spectra of α-synuclein, showing residue-specific enhancements ≥100× over their thermal counterparts. These enhancements, however, vary considerably throughout the residues. The biophysics underlying this residue-specific behavior upon injection of hyperpolarized water is theoretically examined, the information that it carries is compared with results arising from alternative methods, and its overall potential is discussed.

Identification and Rationalization of Kinetic Folding Intermediates for a Low-Density Lipoprotein Receptor Ligand-Binding Module

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time. We demonstrate that misfolded or partially folded disulfide intermediates are indistinguishable from the unfolded state when focusing on the backbone NMR signals, which provide information on the formation of only the final, native state. However, 13C labeling of cysteine side chains differentiated transient intermediates from the unfolded and native states and reported on disulfide bond formation in real time. The cysteine pairings in a dominant intermediate were identified using 13C-edited three-dimensional NMR, and coarse-grained molecular dynamics simulations were used to investigate the preference of this disulfide set over other non-native arrangements. The transient population of LA5 species with particular non-native cysteine connectitivies during folding supports the conclusion that cysteine pairing is not random and that there is a bias toward certain structural ensembles during the folding process, even prior to the binding of calcium.