Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

Ragavan, Mukundan; Iconaru, Luigi I.; Park, Cheon‐Gil; Kriwacki, Richard W.; Hilty, Christian

doi:10.1002/anie.201700464

Cited by 31 publications

(26 citation statements)

References 31 publications

(50 reference statements)

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“…This is, to the best of our knowledge, the lowest concentration achieved by high-resolution NMR spectroscopy in liquids, even relative to other hyperpolarization techniques. For instance, Ragavan et al reported detection of 15 and 20 μM proteins via D-DNP (47,48). This is a considerably higher concentration than the one achieved in the present study.…”

Section: Lc-photo-cidnp Depends Only Weakly On Laser Power At Extremelycontrasting

confidence: 67%

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration

Okuno

Mecha

Yang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Solution-state NMR typically requires 100 μM to 1 mM samples. This limitation prevents applications to mass-limited and aggregationprone target molecules. Photochemically induced dynamic nuclear polarization was adapted to data collection on low-concentration samples by radiofrequency gating, enabling rapid 1D NMR spectral acquisition on aromatic amino acids and proteins bearing aromatic residues at nanomolar concentration, i.e., a full order of magnitude below other hyperpolarization techniques in liquids. Both backbone H 1 -C 13 and side-chain resonances were enhanced, enabling secondary and tertiary structure analysis of proteins with remarkable spectral editing, via the 13 C PREPRINT pulse sequence. Laser-enhanced 2D NMR spectra of 5 μM proteins at 600 MHz display 30-fold better S/N than conventional 2D data collected at 900 MHz. Sensitivity enhancements achieved with this technology, denoted as low-concentration photo-CIDNP (LC-photo-CIDNP), depend only weakly on laser intensity, highlighting the opportunity of safer and more cost-effective hypersensitive NMR applications employing low-power laser sources. hyperpolarization | photo-CIDNP | NMR | proteins | amino acids N MR is an atomic-resolution noninvasive method to probe molecular structure and dynamics. This technique is, however, inherently insensitive due to the unfavorable distribution of nuclear spin states at the near-ambient temperature used in most applications. Methods implemented over the years to overcome the low sensitivity of NMR in liquids include the use of high applied magnetic fields, data acquisition in the time domain followed by Fourier transform, fast data collection schemes, and cryogenic probes (1-3). More recently, nuclear-spin hyperpolarization including Overhauser dynamic nuclear polarization (4, 5), optical pumping (6-9), parahydrogen-induced polarization (10, 11), signal amplification by reversible exchange (12), and dissolution dynamic nuclear polarization (D-DNP) (13-17) have displayed significant potential (3).Despite the above technological advances, typical liquid-state NMR experiments employing hyperpolarization still require very expensive instrumentation; harsh hyperpolarization conditions; long polarization times; and last but not least, ≥50-100 μM sample concentration. In addition, NMR data collection of dilute biomolecules in physiologically relevant milieux is often unfeasible. While some of the above challenges may in principle be overcome by concentrating NMR samples and employing probes accommodating small sample volumes, this process is often unfeasible due to limited amounts of available material or to undesirable aggregation. In summary, there is a compelling need to further enhance the sensitivity of solution-state NMR spectroscopy.Photochemically induced dynamic nuclear polarization (photo-CIDNP) is a spin-selective technique involving the transient generation of radical pairs (Fig. 1A). This methodology has been traditionally employed to gauge macromolecular solventexposure (18)(19)(20). More recently, phot...

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Section: Lc-photo-cidnp Depends Only Weakly On Laser Power At Extremelycontrasting

confidence: 67%

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration

Okuno

Mecha

Yang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…Ragavan et al demonstrated the real‐time observation of 13 C DNP‐enhanced NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds, providing evidence for a structural change of p27 associated with the interaction with Cdk2/cyclin A . Comparing 13 C NMR spectra of hyperpolarized p27 with and without admixing of Cdk2/cyclin A complex, higher signal intensities of 13 CO groups in samples of hyperpolarized p27 mixed with Cdk2/cyclin A complex was observed, which indicated the formation of α‐helical secondary structures.…”

Section: Applicationsmentioning

confidence: 99%

“…Comparing 13 C NMR spectra of hyperpolarized p27 with and without admixing of Cdk2/cyclin A complex, higher signal intensities of 13 CO groups in samples of hyperpolarized p27 mixed with Cdk2/cyclin A complex was observed, which indicated the formation of α‐helical secondary structures. The series of spectra obtained from samples with and without Cdk2/cyclin A complex was analyzed by fitting intensities of spectra at each chemical shift . From the fit, decay rate constants were obtained.…”

Section: Applicationsmentioning

confidence: 99%

Applications of dissolution dynamic nuclear polarization in chemistry and biochemistry

Zhang

Hilty

2018

Magnetic Reson in Chemistry

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Sensitivity of detection is one of the most limiting aspects when applying NMR spectroscopy to current problems in the molecular sciences. A number of hyperpolarization methods exist for increasing the population difference between nuclear spin Zeeman states and enhance the signal-to-noise ratio by orders of magnitude. Among these methods, dissolution dynamic nuclear polarization (D-DNP) is unique in its capability of providing high spin polarization for many types of molecules in the liquid state. Originally proposed for biomedical applications including in vivo imaging, applications in high resolution NMR spectroscopy are now emerging. These applications are the focus of the present review. Using D-DNP, a small sample aliquot is first hyperpolarized as a frozen solid at low temperature, followed by dissolution into the liquid state. D-DNP extends the capabilities of liquid state NMR spectroscopy towards shorter timescales and enables the study of nonequilibrium processes, such as the kinetics and mechanisms of reactions. It allows the determination of intermolecular interactions, in particular based on spin relaxation parameters. At the same time, a challenge in the application of this hyperpolarization method is that spin polarization is nonrenewable. Substantial effort has been devoted to develop methods for enabling rapid correlation spectroscopy, the measurement of time-dependent signals, and the extension of the observable time window. With these methods, D-DNP has the potential to open new application areas in the chemical and biochemical sciences.

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“…This core‐periphery learning hypothesis is novel, since it connects previously fragmented knowledge on the capability of model networks to develop system attractors in learning processes on network periphery‐generated creative innovations and on the differential role of core and periphery nodes in attractor formation in a large variety of real‐world networks . Moreover, the hypothesis expands these lines of evidence to a general adaptation and learning mechanism of complex systems.…”

Section: The Core‐periphery Learning Hypothesis: Known Stimuli Trimentioning

confidence: 90%

“…Disordered segments often fold upon binding to the upstream signaling partner, allowing “conformational signaling” as exemplified by the nuclear hormone receptor family and the cAMP response element‐binding protein (CREB) among many others . Folding of intrinsically disordered segments often occurs in the range of seconds, which is much slower than either the picosec time‐scale of protein core‐mediated signaling, or the nsec to msec timescale of conformational changes …”

Section: Examples Of System Response Duality To Well‐known Versus mentioning

confidence: 99%

The Wisdom of Networks: A General Adaptation and Learning Mechanism of Complex Systems

Csermely

2017

BioEssays

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I hypothesize that re-occurring prior experience of complex systems mobilizes a fast response, whose attractor is encoded by their strongly connected network core. In contrast, responses to novel stimuli are often slow and require the weakly connected network periphery. Upon repeated stimulus, peripheral network nodes remodel the network core that encodes the attractor of the new response. This "core-periphery learning" theory reviews and generalizes the heretofore fragmented knowledge on attractor formation by neural networks, periphery-driven innovation, and a number of recent reports on the adaptation of protein, neuronal, and social networks. The core-periphery learning theory may increase our understanding of signaling, memory formation, information encoding and decision-making processes. Moreover, the power of network periphery-related "wisdom of crowds" inventing creative, novel responses indicates that deliberative democracy is a slow yet efficient learning strategy developed as the success of a billion-year evolution. Also see the video abstract here: https://youtu.be/IIjP7zWGjVE.

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Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

Cited by 31 publications

References 31 publications

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration

Applications of dissolution dynamic nuclear polarization in chemistry and biochemistry

The Wisdom of Networks: A General Adaptation and Learning Mechanism of Complex Systems

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