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Residual dipolar couplings: are multiple independent alignments always possible?

Higman VA, Boyd J, Smith LJ, Redfield C - J. Biomol. NMR (2010)

Bottom Line: Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters.Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR.Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, UK.

ABSTRACT
RDCs for the 14 kDa protein hen egg-white lysozyme (HEWL) have been measured in eight different alignment media. The elongated shape and strongly positively charged surface of HEWL appear to limit the protein to four main alignment orientations. Furthermore, low levels of alignment and the protein's interaction with some alignment media increases the experimental error. Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters. For an analysis of protein dynamics the data must be obtained with very low errors in at least three or five independent alignment media (depending on the method used) and so far, such data have only been reported for three small 6-8 kDa proteins with identical folds: ubiquitin, GB1 and GB3. Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR. Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.

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SECONDA analysis (Hus and Brüschweiler 2002; Hus et al. 2003) for the eight sets of RDCs measured for HEWL. RDCs were normalised prior to the analysis as described by Lakomek et al. (2008) The eigenvalues are plotted in decreasing size and the ratio between the 5th and 6th eigenvalue, ρ, is indicated. The ratio between the 1st and 5th eigenvalues is provided in parentheses. The analysis included all eight data sets (a) or all data sets excluding medium 1 (b), medium 2 (c), medium 3 (d), medium 4 (e), medium 5 (f), medium 6 (g), medium 7 (h) or medium 8 (i)
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Fig2: SECONDA analysis (Hus and Brüschweiler 2002; Hus et al. 2003) for the eight sets of RDCs measured for HEWL. RDCs were normalised prior to the analysis as described by Lakomek et al. (2008) The eigenvalues are plotted in decreasing size and the ratio between the 5th and 6th eigenvalue, ρ, is indicated. The ratio between the 1st and 5th eigenvalues is provided in parentheses. The analysis included all eight data sets (a) or all data sets excluding medium 1 (b), medium 2 (c), medium 3 (d), medium 4 (e), medium 5 (f), medium 6 (g), medium 7 (h) or medium 8 (i)

Mentions: The protein adopts four main alignments (Fig. 1a, axis system 2) dominated by its shape and charge. The elongated shape of HEWL (D///D⊥ = 1.3) is the overriding factor in aligning the protein in both uncharged and positively charged media (media 1–7) and accounts for three of the four main alignments (Fig. 1a, grey, pale blue and pale green in axis system 2). Interestingly, the steric alignment induced by the compressed polyacrylamide gel differs significantly from that observed in the other uncharged and positively charged media, and is not simply related by a factor of −½ (Fig. 1a dark blue/pale green). Although the alignments shown in grey and pale blue in Fig. 1a appear distinct, they are still highly correlated (Table 2) and may not be strictly independent. Alignment in a strongly negatively charged medium, such as Pf1 phage, provides the fourth main alignment (Fig. 1a, purple/black). However, due to the highly positively charged surface of HEWL (pI ≈ 11), the protein strongly interacts with the negatively charged phage and a salt concentration of 500 mM was required in order to obtain even a medium quality spectrum of HEWL (Figure S1). This is undesirable, since the additional salt may influence the structure and/or dynamics of the protein. This highlights a further problem when trying to modulate the alignment tensor with different media: certain media place significant constraints on the physical conditions (temperature, pH, ionic strength). SECONDA analysis (Hus and Brüschweiler 2002) of the HEWL data across all eight media or any sub-group of seven media (Fig. 2) gives rise to a ratio between the 5th and 6th eigenvalue, ρ, of 1.1–3.4. Furthermore, the ratio between the 1st and 5th eigenvalues, an indicator for the signal to noise level, is significantly greater than 1 in all cases. Thus the error level across the different media is not sufficient for information about dynamics to be extracted.Table 2


Residual dipolar couplings: are multiple independent alignments always possible?

Higman VA, Boyd J, Smith LJ, Redfield C - J. Biomol. NMR (2010)

SECONDA analysis (Hus and Brüschweiler 2002; Hus et al. 2003) for the eight sets of RDCs measured for HEWL. RDCs were normalised prior to the analysis as described by Lakomek et al. (2008) The eigenvalues are plotted in decreasing size and the ratio between the 5th and 6th eigenvalue, ρ, is indicated. The ratio between the 1st and 5th eigenvalues is provided in parentheses. The analysis included all eight data sets (a) or all data sets excluding medium 1 (b), medium 2 (c), medium 3 (d), medium 4 (e), medium 5 (f), medium 6 (g), medium 7 (h) or medium 8 (i)
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Related In: Results  -  Collection

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Fig2: SECONDA analysis (Hus and Brüschweiler 2002; Hus et al. 2003) for the eight sets of RDCs measured for HEWL. RDCs were normalised prior to the analysis as described by Lakomek et al. (2008) The eigenvalues are plotted in decreasing size and the ratio between the 5th and 6th eigenvalue, ρ, is indicated. The ratio between the 1st and 5th eigenvalues is provided in parentheses. The analysis included all eight data sets (a) or all data sets excluding medium 1 (b), medium 2 (c), medium 3 (d), medium 4 (e), medium 5 (f), medium 6 (g), medium 7 (h) or medium 8 (i)
Mentions: The protein adopts four main alignments (Fig. 1a, axis system 2) dominated by its shape and charge. The elongated shape of HEWL (D///D⊥ = 1.3) is the overriding factor in aligning the protein in both uncharged and positively charged media (media 1–7) and accounts for three of the four main alignments (Fig. 1a, grey, pale blue and pale green in axis system 2). Interestingly, the steric alignment induced by the compressed polyacrylamide gel differs significantly from that observed in the other uncharged and positively charged media, and is not simply related by a factor of −½ (Fig. 1a dark blue/pale green). Although the alignments shown in grey and pale blue in Fig. 1a appear distinct, they are still highly correlated (Table 2) and may not be strictly independent. Alignment in a strongly negatively charged medium, such as Pf1 phage, provides the fourth main alignment (Fig. 1a, purple/black). However, due to the highly positively charged surface of HEWL (pI ≈ 11), the protein strongly interacts with the negatively charged phage and a salt concentration of 500 mM was required in order to obtain even a medium quality spectrum of HEWL (Figure S1). This is undesirable, since the additional salt may influence the structure and/or dynamics of the protein. This highlights a further problem when trying to modulate the alignment tensor with different media: certain media place significant constraints on the physical conditions (temperature, pH, ionic strength). SECONDA analysis (Hus and Brüschweiler 2002) of the HEWL data across all eight media or any sub-group of seven media (Fig. 2) gives rise to a ratio between the 5th and 6th eigenvalue, ρ, of 1.1–3.4. Furthermore, the ratio between the 1st and 5th eigenvalues, an indicator for the signal to noise level, is significantly greater than 1 in all cases. Thus the error level across the different media is not sufficient for information about dynamics to be extracted.Table 2

Bottom Line: Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters.Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR.Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, UK.

ABSTRACT
RDCs for the 14 kDa protein hen egg-white lysozyme (HEWL) have been measured in eight different alignment media. The elongated shape and strongly positively charged surface of HEWL appear to limit the protein to four main alignment orientations. Furthermore, low levels of alignment and the protein's interaction with some alignment media increases the experimental error. Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters. For an analysis of protein dynamics the data must be obtained with very low errors in at least three or five independent alignment media (depending on the method used) and so far, such data have only been reported for three small 6-8 kDa proteins with identical folds: ubiquitin, GB1 and GB3. Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR. Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.

Show MeSH