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1. Predicted and Experimental NMR Chemical Shifts at Variable Temperatures: The Effect of Protein Conformational Dynamics

   https://doi.org/10.1021/acs.jpclett.3c02589  

   Yi, Xu; Zhang, Lichirui; Friesner, Richard A.; McDermott, Ann (February 2024, The Journal of Physical Chemistry Letters)

   PNAS (Ed.)

   While low-temperature Nuclear Magnetic Resonance (NMR) holds great promise for the analysis of unstable samples and for sensitizing NMR detection, spectral broadening in frozen protein samples is a common experimental challenge. One hypothesis explaining the additional linewidth is that a variety of conformations are in rapid equilibrium at room temperature and become frozen, creating an inhomogeneous distribution at cryogenic temperatures. Here, we investigate conformational heterogeneity by measuring the backbone torsion angle (Ψ) in Escherichia coli Dihydrofolate Reductase (DHFR) at 105 K. Motivated by the particularly broad N chemical shift distribution in this and other examples, we modified an established NCCN Ψ experiment to correlate the chemical shift of Ni+1 to Ψi. With selective 15N and 13C enrichment of Ile, only the unique I60-I61 pair was expected to be detected in 13C’-15N correlation spectrum. For this unique amide, we detected three different conformation basins based on dispersed chemical shifts. Backbone torsion angles Ψ were determined for each basin: 114 ± 7° for the major peak and 150 ± 8° and 164 ± 16° for the minor peaks as contrasted with 118° for the X-ray crystal structure (and 118° to 130° for various previously reported structures). These studies support the hypothesis that inhomogeneous distributions of protein backbone torsion angles contribute to the lineshape broadening in low-temperature NMR spectra. 

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2. Predicted and Experimental NMR Chemical Shifts at Variable Temperatures: The Effect of Protein Conformational Dynamics

   Xu Yi, Lichirui Zhang (January 2023, bioRxiv)

   na (Ed.)

   NMR chemical shifts provide a sensitive probe of protein structure and dynamics. Prediction of shifts, and therefore interpretation of shifts, particularly for the frequently measured amidic 15N sites, remains a tall challenge. We demonstrate that protein 15N chemical shift prediction from QM/MM predictions can be improved if conformational variation is included via MD sampling, focusing on the antibiotic target, E. coli Dihydrofolate reductase (DHFR). Variations of up to 25 ppm in predicted 15N chemical shifts are observed over the trajectory. For solution shifts the average of fluctuations on the low picosecond timescale results in a superior prediction to a single optimal conformation. For low temperature solid state measurements, the histogram of predicted shifts for locally minimized snapshots with specific solvent arrangements sampled from the trajectory explains the heterogeneous linewidths; in other words, the conformations and associated solvent are ‘frozen out’ at low temperatures and result in inhomogeneously broadened NMR peaks. We identified conformational degrees of freedom that contribute to chemical shift variation. Backbone torsion angles show high amplitude fluctuations during the trajectory on the low picosecond timescale. For a number of residues, including I60, ψ varies by up to 60º within a conformational basin during the MD simulations, despite the fact that I60 (and other sites studied) are in a secondary structure element and remain well folded during the trajectory. Fluctuations in ψ appear to be compensated by other degrees of freedom in the protein, including φ of the succeeding residue, resulting in “rocking” of the amide plane with changes in hydrogen bonding interactions. Good agreement for both room temperature and low temperature NMR spectra provides strong support for the specific approach to conformational averaging of computed chemical shifts. 

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3. 1H, 13C, and 15N resonance assignment and secondary structure of the pheromone binding protein from the agricultural pest Ostrinia furnacalis (OfurPBP2)

   https://doi.org/10.1007/s12104-020-09930  

   Dahal, S. (April 2020, Biomolecular NMR assignments)

   Ostrinia furnacalis, a lepidopteran moth, is an invasive pest found in Asia, Australia, Africa and parts of the United States. The Ostrinia furnacalis pheromone-binding protein2 (OfurPBP2), present in the male moth antenna, plays a role in the detection of female-secreted pheromone in a process that leads to mating. To understand the structural mechanism of pheromone binding and release in this pest, we have initiated characterization of OfurPBP2 by solution NMR. Here, we report the backbone resonance assignments and the secondary structural elements of OfurPBP2 at pH 6.5 using uniformly 13C, 15N-labeled protein with various triple resonance NMR experiments. The assignments are 97 % completed for backbone and 88 % completed for side-chain resonances. The secondary structure of OfurPBP2, based on backbone chemical shifts, consists of eight α-helices, including a well-structured C-terminal helix. 

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4. 1H, 15N and 13C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP

   https://doi.org/10.1007/s12104-016-9710-y  

   Zhang, Huaqun; McGlone, Cameron; Mannion, Matthew M.; Page, Richard C. (October 2016, Biomolecular NMR Assignments)

   The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the 1H, 13C, and 15N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90. 

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5. Improving the accuracy of solid-state nuclear magnetic resonance chemical shift prediction with a simple molecular correction

   https://doi.org/10.1039/C9CP01666J  

   Dracinsky, Martin; Unzueta, Pablo; Beran, Gregory J. (January 2019, Physical Chemistry Chemical Physics)

   A fast, straightforward method for computing NMR chemical shieldings of crystalline solids is proposed. The method combines the advantages of both conventional approaches: periodic calculations using plane-wave basis sets and molecular computational approaches. The periodic calculations capture the periodic nature of crystalline solids, but the computational level of the electronic structure calculation is limited to general-gradient-approximation (GGA) density functionals. It is demonstrated that a correction to the GGA result calculated on an isolated molecule at a higher level of theory significantly improves the correlations between experimental and calculated chemical shifts while adding almost no additional computational cost. Corrections calculated with a hybrid density functional improved the accuracy of 13C, 15N and 17O chemical shift predictions significantly and allowed identifying errors in previously published experimental data. Applications of the approach to crystalline isocytosine, methacrylamide, and testosterone are presented. 

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