scholarly journals Protein dynamics insights from <sup>15</sup>N-<sup>1</sup>H (TROSY) HSQC

2021 ◽  
Author(s):  
Erik R. P. Zuiderweg
Keyword(s):  
Conformational Changes ◽  
Conformational Exchange ◽  
Amide Proton ◽  
15N Nmr ◽  
15N Relaxation ◽  
Dynamic Information ◽  
Small Proteins ◽  
Line Widths ◽  
Motional Narrowing ◽  
Exchange Broadening

Abstract. Protein dynamic information is customarily extracted from 15N NMR spin-relaxation experiments. These experiments can only be applied to (small) proteins that can be dissolved to high concentrations. However, most proteins of interest to the biochemical and biomedical community are large and relatively insoluble. These proteins often have functional conformational changes, and it is particularly regretful that these processes cannot be supplemented by dynamical information from NMR. We ask here whether (some) dynamic information can be obtained form the 1H line widths in 15N-1H HSQC spectra. Such spectra are widely available, also for larger proteins. We developed a computer program to predict amide proton line widths from (crystal) structures. As a calibration, we test our approach on BPTI. We find that we can predict most of the distribution of experimental amide proton line widths if we take the dipole-dipole interaction with at least 40 surrounding protons into account. When focusing our attention the outliers of the distribution, we find for BPTI a cluster of conformationally broadened 1HN resonances of residues in strands 10–15 and 36–40 of the beta sheet. Conformational exchange broadening of the 15NH resonances for these residues was previously reported using 15N relaxation measurements (Szyperski et al., J. Biomol. NMR 3, 151–164, 1993). There is little or no evidence for motional narrowing of the 1HN resonances, also in agreement with earlier data using 15N relaxation methods (Beeser et.al, J. Mol. Biol. 269, 154–164, 1997). We also apply our program to 42 kDa domain of the human Hsc70 protein. In this case, there is no previous 15N relaxation data to compare with, but we find, again from the outliers of the distribution, both exchange broadening and motional narrowing that appears to corroborate previous conformational insights for this domain.

scholarly journals Insights into Protein Dynamics from <sup>15</sup>N-<sup>1</sup>H HSQC

2021 ◽  
Author(s):  
Erik R. P. Zuiderweg
Keyword(s):  
Conformational Changes ◽  
Conformational Exchange ◽  
Amide Proton ◽  
15N Nmr ◽  
15N Relaxation ◽  
Relaxation Data ◽  
Dynamic Information ◽  
Small Proteins ◽  
Relaxation Experiments ◽  
Line Widths

Abstract. Protein dynamic information is customarily extracted from 15N NMR spin-relaxation experiments. These experiments can only be applied to (small) proteins that can be dissolved to high concentrations. However, most proteins of interest to the biochemical and biomedical community are large and relatively insoluble. These proteins often have functional conformational changes, and it is particularly regretful that these processes cannot be supplemented by dynamical information from NMR. We ask here whether (some) dynamic information can be obtained from the 1H line widths in 15N-1H HSQC spectra. Such spectra are widely available, also for larger proteins. We developed computer programs to predict amide proton line widths from (crystal) structures. We aim to answer the following basic questions: is the 1H linewidth of a HSQC cross peak smaller than average because its 1H nucleus has few dipolar neighbors, or because the resonance is motionally narrowed? Is a broad line broad because of conformational exchange, or because the 1H nucleus resides in a dense proton environment? We calibrate our programs by comparing computational and experimental results for GB1 (58 residues). We deduce that GB1 has low average 1HN order parameters (0.8), in broad agreement with what was found by others from 15N relaxation experiments (Idiyatullin et al., 2003). We apply the program to the BPTI crystal structure and compare the results with a 15N-1H HSQC spectrum of BPTI (56 residues) and identify a cluster of conformationally broadened 1HN resonances that belong to an area, for which millisecond dynamics has been previously reported from 15N relaxation data (Szyperski et al., J. Biomol. NMR 3, 151-164, 1993). We feel that our computational approach is useful to glean insights into the dynamical properties of larger biomolecules for which high-quality 15N relaxation data cannot be recorded.

scholarly journals In-Situ Flexibility of Both Redox-States of the Chloroplast Regulatory Protein CP12

2021 ◽  
Author(s):  
Helene Launay ◽  
Hui Shao ◽  
Olivier Bornet ◽  
Francois-Xavier Cantrelle ◽  
Regine Lebrun ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Cell Extract ◽  
Proton Exchange ◽  
Conformational Exchange ◽  
Amide Proton ◽  
Cysteine Residues ◽  
Intrinsically Disordered ◽  
Amide Proton Exchange ◽  
C Terminus ◽  
Helical Turn

In the chloroplast, Calvin-Benson-Bassham enzymes are active in the reducing environment imposed in the light via the electrons from the photosystems. In the dark these enzymes are inhibited, and this regulation is mainly mediated via oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges are simultaneous. In oxidized CP12, the C23-C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66-C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment that is, in cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66-C75 pair compared to the C23-C31 pair. Finally, the redox mid-potentials for the two cysteine pairs differ and are similar to those found for phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, that we relate to the regulatory role of CP12.

scholarly journals NMR structure and dynamics of monomeric neutrophil-activating peptide 2

1999 ◽  
Vol 338 (3) ◽  
pp. 591-598 ◽  
Author(s):  
Helen YOUNG ◽  
Vikram ROONGTA ◽  
Thomas J. DALY ◽  
Kevin H. MAYO
Keyword(s):  
Solution Structure ◽  
Computational Modelling ◽  
Terminal Segment ◽  
Conformational Exchange ◽  
15N Nmr ◽  
Model Free ◽  
C Terminus ◽  
Internal Mobility ◽  
Α Helix ◽  
Β Sheet

Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the α-chemokine family. Although NAP-2, like other α-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel β-sheet on which is folded a C-terminal α-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other α-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the β-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from β-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other α-chemokines.

scholarly journals NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 747
Author(s):  
Harri A. Heikkinen ◽  
Sofia M. Backlund ◽  
Hideo Iwaï
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Resonance Assignments ◽  
Nmr Structure ◽  
15N Relaxation ◽  
Nmr Structure Determination ◽  
Nmr Structures ◽  
Small Proteins ◽  
15N Labeling ◽  
Labeling Scheme

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand–protein interaction.

scholarly journals Monitoring Dynamics of Large Membrane Proteins by 19F Paramagnetic Longitudinal Relaxation: Domain Movement in a Glutamate Transporter Homolog

2019 ◽  
Author(s):  
Yun Huang ◽  
Xiaoyu Wang ◽  
Guohua Lv ◽  
Asghar M. Razavi ◽  
Gerard H. M. Huysmans ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Membrane Proteins ◽  
Binding Site ◽  
Conformational Changes ◽  
Glutamate Transporter ◽  
Conformational Exchange ◽  
Substrate Binding Site ◽  
Domain Movement ◽  
Paramagnetic Metal ◽  
Longitudinal Relaxation

AbstractIn proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

scholarly journals Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

2020 ◽  
Vol 117 (6) ◽  
pp. 2938-2947 ◽  
Author(s):  
Reza Amani ◽  
Collin G. Borcik ◽  
Nazmul H. Khan ◽  
Derek B. Versteeg ◽  
Maryam Yekefallah ◽  
...  
Keyword(s):  
Solid State ◽  
Conformational Changes ◽  
Solid State Nmr ◽  
Lipid Binding ◽  
Selectivity Filter ◽  
Conformational Exchange ◽  
Lipid Mixture ◽  
Kir Channels ◽  
Activated State ◽  
Activation Gate

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

Backbone dynamics and amide proton exchange at the two sides of the eremomycin dimer by 15N NMR

2001 ◽  
pp. 501-502 ◽  
Author(s):  
Gy. Batta ◽  
F. Sztaricskai ◽  
M. O. Makarova ◽  
E. G. Gladkikh ◽  
V. V. Pogozheva ◽  
...  
Keyword(s):  
Proton Exchange ◽  
Backbone Dynamics ◽  
Amide Proton ◽  
15N Nmr ◽  
Amide Proton Exchange ◽  
Two Sides
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