Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology

José, A.; Wand, A. Joshua

doi:10.1016/j.ymeth.2018.06.012

Cited by 37 publications

(43 citation statements)

References 116 publications

(132 reference statements)

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“…As internal cavities make a major contribution to the total volume change upon protein structural transition, hydrostatic pressure emerges as a key variable to the study of protein excited states (16). High-pressure (HP) NMR has already profoundly increased our understanding of protein stability, structure, dynamics, and function (24)(25)(26)(27)(28)(29)(30)(31)(32). This relative wealth of information has not, however, led to a consensus picture of the relation of protein thermodynamic stability and structural transitions and its origin, leading to apparently enigmatic descriptions (31)(32)(33)(34).…”

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confidence: 99%

How internal cavities destabilize a protein

Xue

Wakamoto

Kejlberg

et al. 2019

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

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Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified “invisible” folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å−3.

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confidence: 99%

How internal cavities destabilize a protein

Xue

Wakamoto

Kejlberg

et al. 2019

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

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“…It is a safe and reliable system, easy to set up, and suitable for programmable pressure changes including pressure-jump experiments [28]. Technical aspects have recently been reviewed [29]. A similar system was developed by Kalbitzer using a sapphire cell [30].…”

Section: Nmr Methodologymentioning

confidence: 99%

Characterization of low-lying excited states of proteins by high-pressure NMR

Williamson

Kitahara

2019

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Hydrostatic pressure alters the free energy of proteins by a few kJ mol-1 , with the amount depending on their partial molar volumes. Because the folded ground state of a protein contains cavities, it is always a state of large partial molar volume. Therefore pressure always destabilises the ground state and increases the population of partially and completely unfolded states. This is a mild and reversible conformational change, which allows the study of excited states under thermodynamic equilibrium conditions. Many of the excited states studied in this way are functionally relevant; they also seem to be very similar to kinetic folding intermediates, thus suggesting that evolution has made use of the T includes features such as ligand binding, structural change during the catalytic cycle, and dynamic allostery.

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“…Thus, NMR spectroscopy is extensively used in a wide range of applications, including organic chemistry [108], biochemistry, polymer chemistry [122], inorganic chemistry [122], structural biology [52], physics [61,[123][124][125][126][127], biology, and drug discovery [52,128,129]. Through NMR experiments, researchers can study samples in the solid state [130][131][132], gel phase [133][134][135][136], tissue state [137][138][139], gas phase, and solution state [140][141][142][143]; these approaches have been used to investigate molecular structures, concentration levels, and molecular dynamics [144][145][146]. Moreover, the continuous development of NMR experimental methods and NMR machinery, such as dynamic nuclear polarization (DNP) and high-field NMR spectrometers, has continuously enhanced research on the physical and chemical properties of samples [216][217][218].…”

Section: Nmr Spectroscopymentioning

confidence: 99%

Using NMR spectroscopy to investigate the role played by copper in prion diseases

et al. 2020

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Prion diseases are a group of rare neurodegenerative disorders that develop as a result of the conformational conversion of normal prion protein (PrP C) to the disease-associated isoform (PrP Sc). The mechanism that actually causes disease remains unclear. However, the mechanism underlying the conformational transformation of prion protein is partially understood-in particular, there is strong evidence that copper ions play a significant functional role in prion proteins and in their conformational conversion. Various models of the interaction of copper ions with prion proteins have been proposed for the Cu (II)-binding, cell-surface glycoprotein known as prion protein (PrP). Changes in the concentration of copper ions in the brain have been associated with prion diseases and there is strong evidence that copper plays a significant functional role in the conformational conversion of PrP. Nevertheless, because copper ions have been shown to have both a positive and negative effect on prion disease onset, the role played by Cu (II) ions in these diseases remains a topic of debate. Because of the unique properties of paramagnetic Cu (II) ions in the magnetic field, their interactions with PrP can be tracked even at single atom resolution using nuclear magnetic resonance (NMR) spectroscopy. Various NMR approaches have been utilized to study the kinetic, thermodynamic, and structural properties of Cu (II)-PrP interactions. Here, we highlight the different models of copper interactions with PrP with particular focus on studies that use NMR spectroscopy to investigate the role played by copper ions in prion diseases.

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Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology

Cited by 37 publications

References 116 publications

How internal cavities destabilize a protein

How internal cavities destabilize a protein

Characterization of low-lying excited states of proteins by high-pressure NMR

Using NMR spectroscopy to investigate the role played by copper in prion diseases

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