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Self-consistent residual dipolar coupling based model-free analysis for the robust determination of nanosecond to microsecond protein dynamics.

Lakomek NA, Walter KF, Farès C, Lange OF, de Groot BL, Grubmüller H, Brüschweiler R, Munk A, Becker S, Meiler J, Griesinger C - J. Biomol. NMR (2008)

Bottom Line: For ubiquitin, the SCRM analysis yields an average RDC-derived order parameter of the NH vectors <S2(rdc)>0.72 +/- 0.02 compared to <S2(LS)> = 0.778 +/- 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-tau(c) window increases the averaged amplitude of mobility observed in the sub-supra-tau(c) window by about 34%.The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-tau(c) time scale (S2(rdc)(NH) = 0.59 +/- 0.03), while it is inconspicuous (S2(LS)(NH)= 0.82) on the sub-tau(c) as well as on micros-ms relaxation dispersion time scales.The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data.

View Article: PubMed Central - PubMed

Affiliation: Department for NMR-based Structural Biology, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen, Germany.

ABSTRACT
Residual dipolar couplings (RDCs) provide information about the dynamic average orientation of inter-nuclear vectors and amplitudes of motion up to milliseconds. They complement relaxation methods, especially on a time-scale window that we have called supra-tau(c) (tau(c) < supra-tau(c) < 50 micros). Here we present a robust approach called Self-Consistent RDC-based Model-free analysis (SCRM) that delivers RDC-based order parameters-independent of the details of the structure used for alignment tensor calculation-as well as the dynamic average orientation of the inter-nuclear vectors in the protein structure in a self-consistent manner. For ubiquitin, the SCRM analysis yields an average RDC-derived order parameter of the NH vectors 0.72 +/- 0.02 compared to = 0.778 +/- 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-tau(c) window increases the averaged amplitude of mobility observed in the sub-supra-tau(c) window by about 34%. For the beta-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter S2(rdc)(NH) = (0.59, 0.72, 0.59) was extracted. The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-tau(c) time scale (S2(rdc)(NH) = 0.59 +/- 0.03), while it is inconspicuous (S2(LS)(NH)= 0.82) on the sub-tau(c) as well as on micros-ms relaxation dispersion time scales. The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data.

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(a) and (b) show Q-values for back-calculated RDCs using the X-ray structure 1ubi for alignment tensor determination (dashed line) and after 4 SCRM cycles using the fitted dynamic average NH vector orientations (black line) both for (a) D23M and (b) D36M. The fit of inter-nuclear vector orientations and determined alignment tensor to the experimental data is improved significantly: starting from  = 0.178 for D23M and  = 0.193 for D36M on average, the Q-values decrease to  = 0.062 for both D23M and D36M after 4 SCRM-cycles. (c) and (d) same as (a) and (b) but for  instead of Q-values. Starting from  = 0.977 for D23M and  = 0.972 for D36M on average,  improves to  = 0.997 after 4 SCRM cycles. (e) and (f): The inter-nuclear angles  enclosed between the dynamic average NH vector orientations and the NH vectors of the starting X-ray structure 1ubi are shown. The average angular deviation is 6.97° for D23M and 6.87° for D36M. (g) and (h): Same as for e and f, but compared to the 1d3z NMR structures. The average deviation to the NMR structure is 4.84° for D23M and 4.52° for D36M. Thus, the agreement between the derived dynamic average NH vector orientations and the NMR structure is significantly better than for the 1ubi structure
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Fig2: (a) and (b) show Q-values for back-calculated RDCs using the X-ray structure 1ubi for alignment tensor determination (dashed line) and after 4 SCRM cycles using the fitted dynamic average NH vector orientations (black line) both for (a) D23M and (b) D36M. The fit of inter-nuclear vector orientations and determined alignment tensor to the experimental data is improved significantly: starting from = 0.178 for D23M and = 0.193 for D36M on average, the Q-values decrease to = 0.062 for both D23M and D36M after 4 SCRM-cycles. (c) and (d) same as (a) and (b) but for instead of Q-values. Starting from = 0.977 for D23M and = 0.972 for D36M on average, improves to = 0.997 after 4 SCRM cycles. (e) and (f): The inter-nuclear angles enclosed between the dynamic average NH vector orientations and the NH vectors of the starting X-ray structure 1ubi are shown. The average angular deviation is 6.97° for D23M and 6.87° for D36M. (g) and (h): Same as for e and f, but compared to the 1d3z NMR structures. The average deviation to the NMR structure is 4.84° for D23M and 4.52° for D36M. Thus, the agreement between the derived dynamic average NH vector orientations and the NMR structure is significantly better than for the 1ubi structure

Mentions: As described in the Material and methods section, the SCRM method was designed to iteratively improve the accuracy of the alignment tensor determination and to adjust the average inter-nuclear vector orientations, and as a result, to further reduce the static Q-values and increase the Pearson correlation coefficient . Indeed, after already 4 SCRM-cycles, the static Q-values decreased to less than half of the original value with = 0.062 for both D23M and D36M (Fig. 2a, b). Simultaneously, the correlation coefficients increased to = 0.997 on average (Fig. 2c, d). Convergence was attained in already 4 cycles of SCRM after which the inter-nuclear vector orientations were found to deviated by less than 0.5 between consecutive SCRM cycles (Figure S2a and S2b in the Supporting Information). Thus the iterative procedure rapidly improves the fit of the static structure to the RDCs as compared to the input X-ray structure.Fig. 2


Self-consistent residual dipolar coupling based model-free analysis for the robust determination of nanosecond to microsecond protein dynamics.

Lakomek NA, Walter KF, Farès C, Lange OF, de Groot BL, Grubmüller H, Brüschweiler R, Munk A, Becker S, Meiler J, Griesinger C - J. Biomol. NMR (2008)

(a) and (b) show Q-values for back-calculated RDCs using the X-ray structure 1ubi for alignment tensor determination (dashed line) and after 4 SCRM cycles using the fitted dynamic average NH vector orientations (black line) both for (a) D23M and (b) D36M. The fit of inter-nuclear vector orientations and determined alignment tensor to the experimental data is improved significantly: starting from  = 0.178 for D23M and  = 0.193 for D36M on average, the Q-values decrease to  = 0.062 for both D23M and D36M after 4 SCRM-cycles. (c) and (d) same as (a) and (b) but for  instead of Q-values. Starting from  = 0.977 for D23M and  = 0.972 for D36M on average,  improves to  = 0.997 after 4 SCRM cycles. (e) and (f): The inter-nuclear angles  enclosed between the dynamic average NH vector orientations and the NH vectors of the starting X-ray structure 1ubi are shown. The average angular deviation is 6.97° for D23M and 6.87° for D36M. (g) and (h): Same as for e and f, but compared to the 1d3z NMR structures. The average deviation to the NMR structure is 4.84° for D23M and 4.52° for D36M. Thus, the agreement between the derived dynamic average NH vector orientations and the NMR structure is significantly better than for the 1ubi structure
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2480484&req=5

Fig2: (a) and (b) show Q-values for back-calculated RDCs using the X-ray structure 1ubi for alignment tensor determination (dashed line) and after 4 SCRM cycles using the fitted dynamic average NH vector orientations (black line) both for (a) D23M and (b) D36M. The fit of inter-nuclear vector orientations and determined alignment tensor to the experimental data is improved significantly: starting from = 0.178 for D23M and = 0.193 for D36M on average, the Q-values decrease to = 0.062 for both D23M and D36M after 4 SCRM-cycles. (c) and (d) same as (a) and (b) but for instead of Q-values. Starting from = 0.977 for D23M and = 0.972 for D36M on average, improves to = 0.997 after 4 SCRM cycles. (e) and (f): The inter-nuclear angles enclosed between the dynamic average NH vector orientations and the NH vectors of the starting X-ray structure 1ubi are shown. The average angular deviation is 6.97° for D23M and 6.87° for D36M. (g) and (h): Same as for e and f, but compared to the 1d3z NMR structures. The average deviation to the NMR structure is 4.84° for D23M and 4.52° for D36M. Thus, the agreement between the derived dynamic average NH vector orientations and the NMR structure is significantly better than for the 1ubi structure
Mentions: As described in the Material and methods section, the SCRM method was designed to iteratively improve the accuracy of the alignment tensor determination and to adjust the average inter-nuclear vector orientations, and as a result, to further reduce the static Q-values and increase the Pearson correlation coefficient . Indeed, after already 4 SCRM-cycles, the static Q-values decreased to less than half of the original value with = 0.062 for both D23M and D36M (Fig. 2a, b). Simultaneously, the correlation coefficients increased to = 0.997 on average (Fig. 2c, d). Convergence was attained in already 4 cycles of SCRM after which the inter-nuclear vector orientations were found to deviated by less than 0.5 between consecutive SCRM cycles (Figure S2a and S2b in the Supporting Information). Thus the iterative procedure rapidly improves the fit of the static structure to the RDCs as compared to the input X-ray structure.Fig. 2

Bottom Line: For ubiquitin, the SCRM analysis yields an average RDC-derived order parameter of the NH vectors <S2(rdc)>0.72 +/- 0.02 compared to <S2(LS)> = 0.778 +/- 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-tau(c) window increases the averaged amplitude of mobility observed in the sub-supra-tau(c) window by about 34%.The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-tau(c) time scale (S2(rdc)(NH) = 0.59 +/- 0.03), while it is inconspicuous (S2(LS)(NH)= 0.82) on the sub-tau(c) as well as on micros-ms relaxation dispersion time scales.The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data.

View Article: PubMed Central - PubMed

Affiliation: Department for NMR-based Structural Biology, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, Goettingen, Germany.

ABSTRACT
Residual dipolar couplings (RDCs) provide information about the dynamic average orientation of inter-nuclear vectors and amplitudes of motion up to milliseconds. They complement relaxation methods, especially on a time-scale window that we have called supra-tau(c) (tau(c) < supra-tau(c) < 50 micros). Here we present a robust approach called Self-Consistent RDC-based Model-free analysis (SCRM) that delivers RDC-based order parameters-independent of the details of the structure used for alignment tensor calculation-as well as the dynamic average orientation of the inter-nuclear vectors in the protein structure in a self-consistent manner. For ubiquitin, the SCRM analysis yields an average RDC-derived order parameter of the NH vectors 0.72 +/- 0.02 compared to = 0.778 +/- 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-tau(c) window increases the averaged amplitude of mobility observed in the sub-supra-tau(c) window by about 34%. For the beta-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter S2(rdc)(NH) = (0.59, 0.72, 0.59) was extracted. The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-tau(c) time scale (S2(rdc)(NH) = 0.59 +/- 0.03), while it is inconspicuous (S2(LS)(NH)= 0.82) on the sub-tau(c) as well as on micros-ms relaxation dispersion time scales. The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data.

Show MeSH