Data from: Gel-based NMR method for observing submicrosecond protein dynamics at atomic resolution
Data files
May 20, 2026 version files 45.68 KB
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Im7_compressed_PAG.csv
8.18 KB
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Im7_uncompressed_PAG.csv
5.65 KB
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KRas_compressed_PAG.csv
10.91 KB
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README.md
4.19 KB
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ubiquitin_agarose.csv
4.70 KB
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ubiquitin_compressed_PAG.csv
7.34 KB
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ubiquitin_uncompressed_PAG.csv
4.71 KB
Abstract
NMR spectroscopy is uniquely suitable of observing functionally important protein motions at atomic resolution under near-physiological conditions in solution. Longitudinal and transverse spin relaxation experiments report about fast subnanosecond motions and low nanosecond motions, but they are insensitive to slower motions. The recently introduced nanoparticle-assisted spin relaxation (NASR) method increases the observation window into the submicrosecond range by measuring the increase in transverse relaxation in the presence of silica nanoparticles. It is demonstrated here how a similar effect can be observed via the transverse relaxation enhancement ∆R2 due to the presence of polyacrylamide and agarose gel. While compressed or stretched polyacrylamide gels are commonly used in protein NMR for residual dipolar coupling measurements, the gel-induced transverse relaxation enhancement, both for compressed and uncompressed gel, directly provides complementary dynamics information with the change in R2 proportional to the site-specific model-free S2 order parameter encompassing dynamics on the sub-microsecond range. This generalized NASR approach is demonstrated for K-Ras and other proteins exhibiting internal dynamics with variable amplitudes on a wide range of timescales.
Description of the data and file structure
- .csv files containing relaxation rates measured in the presence and absence of PAG gels, along with the derived gNASR order parameters and their uncertainties.
- The protein identity and gel condition are specified in the file name.
Files and variables
File：KRas_compressed_PAG.csv
Samples
K-Ras·GDP in the presence and absence of vertically compressed 1.8 cm long 7% PAG gel
Variables
- residue number and amino acid type: primary sequence of the protein
- R1free, R1free (err), R2free, R2free (err): relaxation rates with corresponding uncertainties measured in the absence of gel
- R1gel, R1gel (err), R2gel, R2gel (err): relaxation rates with corresponding uncertainties measured in the presence of gel
- S2(gNASR), S2(gNASR) (err): gNASR order parameters and their uncertainties
File：Im7_compressed_PAG.csv
Samples
Im7 in the presence and absence of vertically compressed 1.8 cm long 7% PAG gel
Variables
- residue number and amino acid type: primary sequence of the protein
- R1free, R1free (err), R2free, R2free (err), ηfree, ηfree (err): relaxation rates with corresponding uncertainties measured in the absence of gel
- R1gel, R1gel (err), R2gel, R2gel (err), ηgel, ηgel (err): relaxation rates with corresponding uncertainties measured in the presence of gel
- S2(gNASR), S2(gNASR) (err), S2η(gNASR), S2η(gNASR) (err): gNASR order parameters and their uncertainties
File：Im7_uncompressed_PAG.csv
Samples
Im7 in the presence and absence of uncompressed 7% PAG gel
Variables
- residue number and amino acid type: primary sequence of the protein
- R1free, R1free (err), R2free, R2free (err): relaxation rates with corresponding uncertainties measured in the absence of gel
- R1gel, R1gel (err), R2gel, R2gel (err): relaxation rates with corresponding uncertainties measured in the presence of gel
- S2(gNASR), S2(gNASR) (err): gNASR order parameters and their uncertainties
File：ubiquitin_compressed_PAG.csv
Samples
Ubiquitin in the presence and absence of vertically compressed 1.8 cm long 7% PAG gel
Variables
- residue number and amino acid type: primary sequence of the protein
- R1free, R1free (err), R2free, R2free (err), ηfree, ηfree (err): relaxation rates with corresponding uncertainties measured in the absence of gel
- R1gel, R1gel (err), R2gel, R2gel (err), ηgel, ηgel (err): relaxation rates with corresponding uncertainties measured in the presence of gel
- S2(gNASR), S2(gNASR) (err), S2η(gNASR), S2η(gNASR) (err): gNASR order parameters and their uncertainties
File：ubiquitin_uncompressed_PAG.csv
Samples
Ubiquitin in the presence and absence of uncompressed 7% PAG gel
Variables
- residue number and amino acid type: primary sequence of the protein
- R1free, R1free (err), R2free, R2free (err): relaxation rates with corresponding uncertainties measured in the absence of gel
- R1gel, R1gel (err), R2gel, R2gel (err): relaxation rates with corresponding uncertainties measured in the presence of gel
- S2(gNASR), S2(gNASR) (err): gNASR order parameters and their uncertainties
File：ubiquitin_agarose.csv
Samples
Ubiquitin in the presence and absence of 2% agarose gel
Variables
- residue number and amino acid type: primary sequence of the protein
- R1free, R1free (err), R2free, R2free (err): relaxation rates with corresponding uncertainties measured in the absence of gel
- R1gel, R1gel (err), R2gel, R2gel (err): relaxation rates with corresponding uncertainties measured in the presence of gel
- S2(gNASR), S2(gNASR) (err): gNASR order parameters and their uncertainties
NMR experiments were performed at 298 K on a Bruker AVANCE III spectrometer operating at 850 MHz 1H resonance frequency (19.97 T) equipped with a TCI cryoprobe. For each protein, backbone amide 15N R1 and R2 spin relaxation rates both in the presence and absence of 7% polyacrylamide gel were obtained from standard R1 and R1ρ relaxation experiments with the recovery delay set to 2 s and a R1ρ spin-lock field strength of 2000 Hz. The resulting R1 and R1ρ relaxation rates were combined to obtain site-specific transverse R2 relaxation rates in standard fashion.
The enhancements in 15N-R2 due to the presence of the gel were then determined for each resonance as ∆R2 = R2gel - R2free. ∆R1 = R1gel – R1free was also determined, providing complementary information about the effective viscosity increase experienced by the protein in the presence of the gel. Furthermore, backbone amide 15N/15N-1H CSA/DD transverse cross-correlation rates ηxy were measured with a recovery delay of 1.5 s and a relaxation period T set between 50 and 80 ms. Analogous to ∆R2, ηxy increases in the presence of the gel, with the enhancements determined for each 15N resonance as ∆ηxy = ηxygel – ηxyfree.
The experimentally determined changes ∆in R2 were converted to S2(gNASR) order parameters by uniform scaling with a prefactor c, S2(gNASR) = c.∆R2, so that the most stable sections of secondary structural elements, namely the centers of the helices and b-sheets, have an average S2(gNASR) = 0.85. The cross-correlation rates ∆ηxy were converted to cross-correlated (gNASR) order parameters in a fully analogous manner.
Errors were estimated from replicate delays and propagated through the analysis using analytical error propagation or Monte Carlo simulations.