Helix mobility and recognition function of the rat thyroid transcription factor homeodomain – hints from 15N-NMR relaxation studies Devrim Gumral, Luana Nadalin, Alessandra Corazza, Federico Fogolari, Giuseppe Damante, ă Paolo Viglino and Gennaro Esposito ` Dipartimento di Scienze e Tecnologie Biomediche, Universita di Udine, Italy Keywords backbone dynamics; model-free approach; NMR 15N relaxation; spectral density mapping; thyroid transcription factor homeodomain Correspondence G Esposito, Dipartimento di Scienze e ` Tecnologie Biomediche, Universita di Udine, P.le Kolbe, 4, 33100 Udine, Italy Fax: +39 0432494301 Tel: +39 0432494321 E-mail: gesposito@mail.dstb.uniud.it (Received 20 October 2007, revised 25 November 2007, accepted 28 November 2007) doi:10.1111/j.1742-4658.2007.06208.x The backbone dynamics of the 15N-labeled homeodomain of the rat thyroid transcription factor has been studied by 2D NMR spectroscopy Longitudinal (R1) and transverse (R2) 15N relaxation rate constants and steady-state {1H}–15N NOEs were measured at 11.7 T These data were analyzed by both the model-free formalism and the reduced spectral density mapping (RSDM) approaches The global rotational correlation time, sm, of the thyroid transcription factor homeodomain in aqueous solution at 286 K was found to be 10.51 ± 0.05 ns by model-free formalism and 9.85 ± 1.79 ns by RSDM calculation The homogeneity of the values of the overall correlation time calculated from the individual (R2 ⁄ R1) ratios suggested a good degree of isotropy of the global molecular motion, consistent with the similar global sm results obtained with the two different methods Tyr25 was found to undergo slow conformational exchange by both methods, whereas this contribution was identified also for Lys21, Gln22, Ile38 and His52 only by RSDM With both methods, the C-terminal fragment of helix III was found to be more flexible than the preceding N-terminal portion, with slightly different limits between rigid and mobile moieties Additionally, Arg53 appeared to be characterized by an intermediate motional freedom between the very flexible N-terminal and C-terminal residues and the structured core of the molecule, suggesting the occurrence of a hinge point Finally, slow-time-scale motions observed at the end of helix I, at the end of helix II and within helix III appear to be consistent with typical fraying transitions at helical C-termini Homeodomains (HDs) comprise a very well-known class of DNA-binding domains occurring in a large family of transcription activators involved in the determination of cell development [1–3] The tertiary structure of the HD of rat thyroid transcription factor (TTF-1), a 67-residue domain, was determined by NMR spectroscopy [4] (Brookhaven Protein Data Bank ID code 1FTT) The whole TTF-1 protein (378 residues) is responsible for transcriptional activa- tion of genes expressed only in follicular thyroid cells [5] and lung epithelial cells [6] The structural features of the TTF-1 HD are the typical ones observed in HDs, i.e three helices (Gln10–Gln22, Ala28–Ile38, Thr43–Gln59) connected by a loose loop (Gln23– Ser27) between helix I and helix II and by a tight turn (His39–Pro42) between helix II and helix III (helix–turn–helix motif; Fig 1) The DNA recognition helix (helix III) is fairly ordered also in the Abbreviations Antp, Antennapedia; HD, homeodomain; MD, molecular dynamics; MF, model-free; RSDM, reduced spectral density mapping; TTF-1, thyroid transcription factor FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 435 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ă Fig Cartoon of the TTF-1 HD backbone (Protein Data Bank code 1FTT) [4] with helix I (brown), helix II (magenta), and the DNA recognition helix, helix III (green) The side chains of the residues whose 15N–1H vectors undergo slow motions, i.e ls-to-ms (Lys21 and Gln22 in helix I, Tyr25 in the large loop, Ile38 in helix II, and His52 in helix III), are in blue, whereas, for Leu26, the red color indicates the coupling of low-frequency and high-frequency dynamics With the exception of His52, all the mentioned residues are located in the hydrophobic core of the molecule (i.e Ile38) or close to residues of this core (i.e Lys21 and Gln22, neighboring Phe20; Tyr25, neighboring Leu26) The drawing was prepared using OpenSource PYMOL (DeLano Scientific LLC, South San Francisco, CA, USA) absence of DNA, as first reported for Antennapedia (Antp) HD [7] For the TTF-1 HD, a discontinuity of the hydrogen bond network between N-terminal and C-terminal moieties of the recognition helix was observed at the highly conserved fragment Asn51– His52–Arg53 [4], suggesting the occurrence of either a kinking or tightening of the local geometry A similar discontinuity had been noted in solution also in the Antp HD [8,9] and the Antp (C39S) HD [10], and indeed, originally, the C-terminal extension of helix III, i.e residues 53–59, was proposed to form helix IV However, in the absence of direct evidence supporting a structural interruption of the geometry of the recognition helix for either Antp or the TTF-1 HD, the anomalous amide exchange pattern and the NOE connectivity data of the C-terminal portion of helix III had to be ascribed to local mobility effects [4,10] Subsequently, a quantitative analysis of H–2H exchange rates of the TTF-1 HD revealed opposite effects to helix III stability within the fragment 51–53 that may be relevant to the conformational dynamics of the HD recognition helix upon DNA binding [11] 436 In the following, we present a 15N-NMR relaxation study of the rat TTF-1 HD to address the backbone dynamics in solution 15N-NMR as well as 13C-NMR relaxation studies can be usefully applied to determine the dynamics of proteins [12,13] In high magnetic fields, the relaxation of these nuclei is mainly governed by dipole–dipole and chemical shift anisotropy mechanisms For globular proteins, the analysis of the experimental relaxation data by means of the model-free (MF) approach [14,15] provides a description of the motions in terms of global overall rotational correlation time, sm, a generalized order parameter, S2, and an effective internal correlation time, se For 15N relaxation data, the generalized order parameter reflects the amplitude of the internal motion of the 15N–1H vectors in the fast ps-to-ns time range An alternative method established to examine 15N–1H vector mobility is based on the estimation and interpretation of the spectral density values from the individual relaxation rates [16–22], an approach most commonly applied in a restricted version referred to as reduced spectral density mapping (RSDM) This method provides an analysis of protein dynamics that requires no model assumptions It gives spectral density values at J(0), J(xN) and J(), directly calculated from the measured relaxation parameters, that contain contributions from the overall as well as the local dynamics Graphical analysis of the spectral density values provides a qualitative picture of the internal motions with no bias, as the whole approach does not make any assumption about the motions to be investigated Results Relaxation parameters The individual R1, R2 and NOE values of the backbone amide 15N nuclei of the TTF-1 HD at 286 K are given in supplementary Table S1, Table S2 and Fig S1 Side-chain nitrogens were not considered for analysis, except for the indole nitrogen of Trp48, which represents a convenient probe with which to monitor the dynamics of the HD hydrophobic core (supplementary Table S1) The longitudinal relaxation rates range between 1.15 and 1.97 s)1 The lowest R1 values are observed for Lys24 and Met37 and the residues of the flexible terminal segments, with a characteristic pattern of decreasing values on approaching these latter segments from the respective adjacent helices The highest R1 values are observed for Ser27, Arg31, Glu32, Ser36, Ile38, Val45, and Trp48 The transverse relaxation rate values, higher than the corresponding R1 constants by FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS D Gumral et al ă one order of magnitude, fall in the range 8.41– 16.53 s)1 The lowest R2 values are shown by the N-terminal and C-terminal residues and by Leu34, Gln44, Arg53, Arg58 and Gln59 A unique value of 26.39 s)1, by far the highest one, is observed for Tyr25, which strongly suggests the presence of a local, low-frequency conformational exchange contribution The steady-state {1H}–15N NOEs span the interval )1.70 ⁄ +0.89 Negative values are observed for the terminal fragments, i.e Arg1–Leu7 and Lys61–Gln67, reflecting the local dynamics characterized by fast motions In particular, the sign inversion transitions of NOEs, seen on approaching the helical tracts from flexible terminal residues, parallel the similar trends observed for relaxation rates, and reflect consistently the changes in local motional properties In the recognition helix, lower NOE values are obtained for the C-terminal moiety, confirming that it is more flexible than the N-terminal one The highest {1H}–15N NOEs were measured for Glu17 in helix I, Ser27 and Leu34 in helix II, and Lys46 and Gln50 in the N-terminal portion of helix III For an isotropically tumbling globular molecule, in the absence of internal motions and with relaxation due to dipole–dipole and chemical shift anisotropy mechanisms, {1H}–15N NOEs can be 1, where sm is the global overand +0.82, for xNsm> all rotational correlation time [13] Within the estimated uncertainty, the residues that show a {1H}–15N NOE higher than the theoretically estimated maximum are Glu17, Leu34 and Lys46 This is conceivably a consequence of the overlap affecting the corresponding resonance Therefore, the experimental data of these three residues were not further considered for subsequent MF and RSDM analysis calculations However, the qualitative implication of a high {1H}–15N NOE for Glu17, Leu34 and Lys46, i.e low specific mobility, is consistent with the NOE trend of the corresponding adjacent residues and hence does not conflict with the global interpretation of the data With the exclusion of the N-terminal octapeptidyl and C-terminal nonapeptidyl fragments of Glu17, Leu34 and Lys46, the average of the {1H}–15N NOEs is 0.68 ± 0.10 (supplementary Table S2) This value can be reliably considered to be the average NOE over the structured core of the investigated TTF-1 HD molecule Backbone dynamics of the rat TTF-1 homeodomain Fig Bar graphs of overall rotational correlation time, smi (ns), generalized order parameter, S2 and effective internal correlation time, se (ps) values as a function of the TTF-1 HD sequence The parameters were obtained from measurements at 11.7 T and 286 K se and S2 values were not calculated for Glu17, Leu34 and Lys46, as their NOE signals exhibited almost 100% overlap Additional blank slots in the correspondence of residues 29 and 42 are for prolines The se values of Ser27 and Gln50 are not reported, because they were not optimized by MF analysis The extension of TTF-1 HD helical segments is depicted above the graphs MF motional parameters Figure shows the individual overall rotational correlation time, smi, calculated from the individual residue R2 ⁄ R1 ratios, the generalized order parameters, S2, and the effective correlation times, se, of the TFF-1 HD from MF analysis of the 15N relaxation parameters at 11.7 T and 286 K with the corresponding uncertainties (the actual values are listed in supplementary Table S3) No other exchanging contributions, Rex, but FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 437 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ă that expected for Tyr25 (14.67 ± 2.35 ns) were found from MF formalism calculations Rotational correlation time From the estimates of smi based on the individual relaxation rate ratios (Fig 2), an average value of 9.7 ± 0.4 ns is extracted for the overall tumbling by considering only the parameters from the best defined (and conceivably most rigid) regions of the TTF-1 HD (Gln10–Gln22, Ala28–Ile38, Thr43–Gln50) as determined from the NMR structure of the molecule [4] When averaging is extended over the whole smi dataset, only a slight difference is obtained, i.e Æsmiæ = 9.5 ± 0.9 ns The excellent agreement between the averages shows that the local segmental mobility differences, albeit remarkable as inferred from the NOE data, have little effect on the value of the Ỉsmiỉ estimate, and adds confidence to the assumption of isotropic motion adopted by the equation of the relaxation rate ratio [13] All the individual smi values lie within 2r from average (95% confidence level), except for Tyr25, due to the high value of the corresponding R2 constant, which is affected by a slow exchange contribution A more accurate estimate of the global sm, obtained by unbiased grid search optimization over the experimental parameters and subsequent Brent minimization [23], in the context of MF calculations, gave a value of 10.51 ± 0.05 ns, i.e slightly higher but not far from the value computed from relaxation rate ratios Local generalized order parameters and internal effective correlation times Besides the optimization of the molecular tumbling rate, MF analysis of relaxation data provides a set of optimized parameters describing local motions Except for Tyr25, all the 15N relaxation data of the TTF-1 HD were satisfactorily fitted by means of a dualmotion model entailing a single-frequency local fluctuation superimposed on the global motion The quality of the fitting was statistically validated by v2 test against the corresponding parameter distribution of Monte Carlo simulations The individual generalized order parameters and internal effective correlation times are plotted in Fig Their values reflect, respectively, the specific amplitude and the frequency of the local fluctuations for the motion of each considered internuclear 15N–1H vector The lowest S2 values and, correspondingly, the shortest se values are obtained at the N-terminal and C-terminal fragments 1–7 and 60–67 of the TTF-1 HD This pattern suggests wide 438 motional freedom of the 15N–1H vectors, which is in line with the disordered NMR structure observed for the same regions [4] The N-terminal flexibility starts to quench before reaching helix I, at Phe8 and Ser9, where both parameters of local backbone dynamics increase This progressive transition pattern is attributed to the involvement of Ser9 in the N-capping motif of helix I [4] The trend of the effective internal correlation time, se (referred to as local correlation time), along helix I features a behavior that appears typical within the whole set of MF-based parameters obtained for the TTF-1 HD, namely an increase of local correlation time with increasing generalized order parameter This behavior is intriguing when compared to the established expectation that associates limited local motional amplitudes, i.e S2 between 0.8 and 1, with fast local motions, i.e small se, and, conversely, wide local motional amplitude, i.e S2 < 0.8, with slow local motion, i.e large se In other words, most often for the TTF-1 HD, S2 and se exhibit an opposite correlation from what is expected This casts substantial doubts on the reliability of the picture emerging from the application of MF formalism to TTF-1 HD relaxation data In detail, the highest S2 values are obtained for Gln50 and Tyr54, two residues that are essential for the DNA recognition specificity of the TTF-1 HD [24,25] The restriction in local motion amplitude, implied by the values of S2, seems consistent with the role of Gln50 and Tyr54, but the corresponding se values are not easily rationalized For Gln50, the optimization procedure fails to fit the experimental data with se £ 11 000 ps A low frequency of the internal motions could be considered to match the above-mentioned correlation between high S2 and large se values In contrast, for Tyr54 a very low value of the optimized se (296 ± 192 ps) is obtained, which is difficult to reconcile with the pattern most commonly observed in the dataset, when S2 is close to The physical picture for Tyr54 becomes consistent with local fluctuations with remarkably limited amplitude and high frequency The high level of uncertainty affecting se of Tyr54 may suggest that the result should be considered as a numeric artefact of the optimization However, a decreasing trend of the internal se coupled with a similar behavior of the generalized order parameter unequivocally emerges on examination of segment 50–55 of the TTF-1 HD (Fig 2) Besides Gln50, optimization fails to retrieve a se £ 11 000 ps also for the data of Ser27, a residue of the loop between helix I and helix II In this case, however, the generalized order parameter is, within the estimated error, lower (0.755 ± 0.072) than the average value observed in the structured molecular core (0.86–0.87) Although the FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS D Gumral et al ă Backbone dynamics of the rat TTF-1 homeodomain Table Mean values and corresponding standard deviations (in parentheses) of S2 (dimensionless) and se (ps) parameters for the secondary structure elements of the TTF-1 HD at 286 K Structural unit ỈS2ỉ Helix I (10–22) Helix II (28–38) Helix III (43–59) Helix III (43–52) Helix III (53–59) Helix III (42–56) Helix III (51–56) Loop (23–27) Tight turn (39–42) N-terminus (1–9) C-terminus (60–67) 0.87 0.86 0.85 0.87 0.82 0.87 0.86 0.84 0.92 0.63 0.58 Ỉswa Ỉs (0.04) (0.04) (0.08) (0.06) (0.09) (0.06) (0.06) (0.08) (0.04) (0.19) (0.09) 1983 2493 1486 1885 1030 1630 1345 1038 1960 805 514 (406) (1374) (780) (710) (613) (809) (740) (542) (1047) (752) (277) 1964 1561 1008 1596 905 1297 1142 468 1403 276 206 (37) (57) (19) (51) (21) (37) (47) (47) (106) (3) (3) a Weighted average calculated using the individual se uncertainties (ri) as weighting factors (1 ⁄ ri2) large error in S2 may reflect some problems with the available data quality, a reduced motional rate for the Ser27 backbone appears to be plausible, considering its involvement in the defective capping of helix II [4] At this stage, the results are better described by considering the average values observed in the different secondary structure elements as reported in Table The local dynamics of the three helical regions of the TTF-1 HD look very similar when only the average generalized order parameters are considered A clear difference emerges, however, if the internal correlation times are taken into account Only the motion of helix I appears quite uniform, as inferred from the similar values of the mean and weighed mean se Helix II shows the largest variability in local fluctuation frequency, despite the fact that the relative mean generalized order parameter and the standard deviation are very close to the corresponding counterparts of helix I This result can be rationalized on a structural basis Helix II, in fact, should be the least stable among the TTF-1 HD helices, because of its incomplete hydrogen bond network, which is due to defective N-capping and distortions introduced by Pro29 At the same time, the side chains of residues 34, 35 and 38 are tightly anchored in the hydrophobic core of the molecule, whereas the Glu30 side chain is involved in a salt bridge [4] The restricted mobility of four side chains, out of 10 in helix II, appears to be coupled to a lower motional frequency of the corresponding or adjacent backbone amide bond vectors, which accounts for the inhomogeneity of the local correlation times For helix III, the inhomogeneity can be easily appreciated by inspecting Fig 2, where the well-known difference between the N-terminal and C-terminal moieties of the recognition helix can be seen If the MF parameter averages of Table are accordingly split into average values for segments 43–52 and 53– 59, some internal motion inhomogeneity of helix III is seen to occur also within the single fragments The N-terminal portion exhibits slightly higher and standard deviation than helix I and helix II, and a broad distribution of se, with a weighted average around 1.5 ns, like helix II Again as with helix II, some side chains in this part of the recognition helix (residues 45, 48 and 49) contribute to the hydrophobic core of the molecule Thus, hydrophobic core anchoring has similar results for internal fluctuations in helix II and the N-terminal moiety of helix III Overall, it seems that the whole motional regime of the TTF-1 HD, in the experimental conditions chosen for obtaining the relaxation data (286 K), matches only poorly (and qualitatively) the behavior needed to comply with the implicit conditions imposed by the MF approach In most cases, an increase ⁄ decrease in the generalized order parameter corresponds to an increased ⁄ decreased se, which calls for a motional regime that appears to be inconsistent within the MF framework However, all attempts to fit the experimental data with the extended MF approach [26], which uses a double-timescale model for internal motions, were also unsuccessful It is tempting to speculate that the physically puzzling picture emerging from the MFbased fitting of the majority of the TTF-1 HD relaxation data could be attributed to correlated local dynamics that occur on a timescale similar to that of the overall tumbling Graphical analysis of spectral densities Spectral densities at three frequencies [J(0), J(xN) and J(0.87xH)] were calculated according to the matrix equation given in supplementary Doc S1 The individual spectral density values along the sequence of the TTF-1 HD are displayed in the bar graphs of Fig 3, and the corresponding numerical values are given in supplementary Table S4 Linear correlations between J(0) and J(xN) and between J(0) and J(0.87xH) for the ` TTF-1 HD were then examined as proposed by Lefevre et al [21] The fit was obtained by linear regression, and only the corresponding J(0)–J(xN) correlation plot is shown in Fig The localization of the experimental points in Fig along the correlation line is directly related to the distribution of the energy between the overall tumbling and the internal mobility, and is indicative of the degree of internal restraint of each 15N–1H vector motion In Fig 4, most of the points cluster in the same region The dashed curve, called the theoretical FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 439 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ¨ Fig Bar graphs of spectral density function values (ns) at the zero, xN and 0.87xH frequencies, versus the sequence of the TTF-1 HD Measurements were done at 11.7 T and 286 K Blank slots are for residues 29 and 42 (prolines) and Glu17, Leu34 and Lys46, which were excluded because of the extensive overlap affecting the corresponding signals Correlations were calculated by means of MATHEMATICA 5.2 software, using the relaxation dataset given in supplementary Table S2 Relaxation data obtained from linear prediction were used for calculation only when the error introduced by the procedure was acceptable, as discussed in supplementary Doc S1 The extension of TTF-1 HD helical segments is depicted above the graphs curve, indicates the spectral density values expected for a simple Lorentzian model of J(x) calculated over a very wide range of correlation times, s Most of the 440 Fig J(xN)–J(0) correlation for the individual residues of the TTF1 HD from 15N relaxation measurements Different colors are used to indicate the distinct groups of residues along the sequence, i.e N-terminal (orange), C-terminal (violet), helix I (yellow), helix II (pink), helix III (green), loop (cyan), tight turn (brown), and residues that undergo conformational exchange motions (blue) The fit (dark solid line) was obtained by linear regression with the exclusion of Arg1 and Gln67 (which exhibit strong negative NOE values) and Lys21, Gln22, Tyr25, Ile38 and His52 [which make conformational exchange contributions to J(0)] The dashed curve (theoretical curve) was calculated for J(0) and J(xN) as a function of s, using a simple Lorentzian function The left-hand inset shows an overview of the theoretical curve and the fitting line to highlight the two intercept points The right-hand inset shows Tyr25 correlation, which occurs outside the plotted area Analytically, J(0.87xH) depends only on the cross-relaxation rate; that is, it is largely determined by the heteronuclear NOE and thus it is most sensitive to high-frequency motions of the protein backbone On the other hand, the value of J(xN) is extracted also from R1, whereas J(0) is determined also by both R1 and R2 Therefore, J(0) is sensitive to both nanosecond timescale motions and contributions from ls-toms slow exchange processes For this reason, the main information on dynamics can be derived from analysis of J(0) A plot of the correlation J(0.87xH)–J(0) is given in supplementary Fig S2 experimental points accumulate rather close to the upper intercept of the theoretical curve and the fitting (solid) line, where the motion of a unique 15N–1H vector is defined by a single Lorentzian function with a global overall rotational correlation time, sm The points from N-terminal and C-terminal residues (Arg1–Leu7 and Ala60–Gln67), together with those from Arg58 and Gln59 in the C-terminal end of helix III, are located apart from the major cluster, towards the lower intercept of the theoretical curve and fitting line (Fig 4, left inset), where the motion of FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ê 2007 FEBS D Gumral et al ă a unique 15N–1H vector is defined by a single Lorentzian function with a fast s that is interpreted as generalized internal correlation time, sgi For any point between the upper and lower intercepts of the theoretical curve with the fitting line, the spectral density function can be expressed as a linear combination of the two Lorentzian functions defined by sm and sgi, respectively The proximity to one of the intercepts between the theoretical and fitting curves reflects the relative contribution of each component Lorentzian function to the specific spectral density of each experimental point Therefore, according to the RSDM analysis [21], most of the 15N–1H vectors of the TTF-1 HD core move at the rate of the overall rotational correlation frequency, and relaxation mainly occurs as a result of overall rotational diffusion Among all the TTF-1 HD backbone 15N–1H vectors, those from disordered N-terminal and C-terminal residues, together with Arg58 and Gln59, are the most mobile ones and exhibit fast-timescale (ps-to-ns) motion In Fig 4, the points corresponding to residues Lys21, Gln22, Tyr25, Ile38 and His52 are shifted to the right above the theoretical line, which is a typical pattern for the occurrence of a slow (ls-to-ms) exchange process The data relative to Lys24 and Met37, together with those of several terminal residues (Arg1, Arg2, Ala64, Gln66 and Gln67), fall outside the major cluster of points and feature a distinct dynamic behavior as compared to the remaining 15N–1H vectors of the core Their spectral density functions cannot be expressed with only two Lorentzian functions In the tightening ⁄ kink of the recognition helix introduced by the Asn51–His52–Arg53 tripeptide, His52 and Arg53 show rather different dynamic behaviors Arg53 appears to possess an intermediate motional freedom between those of the N-terminal and C-terminal residues and the core; that is, it undergoes ps-to-ns timescale motion On the other hand, His52 shows slow conformational exchange contributions in the ls-to-ms timescale, as mentioned above A similar situation is observed for the pairs Glu30–Arg31 and Gln44–Val45, with the first residues exibiting faster motions (ps-to-ns timescale), and the latter residues slower motions on the nanosecond timescale Detailed analysis of the spectral density functions can be performed using the bar charts of Fig to obtain the individual dynamic properties of each 15 N–1H vector It can be seen that the 15N–1H vectors of the N-terminal and C-terminal residues undergo the most rapid motions as compared to the rest of the TTF-1 HD backbone This is highlighted by low J(0) Backbone dynamics of the rat TTF-1 homeodomain and J(xN) values and correspondingly high J(0.87xH) values, a pattern that is typically expected when the considered internuclear vectors reorient on a fast (psto-ns) timescale In the loop between helix I and helix II, Tyr25 shows a J(0) value that is much higher than that of any other 15N–1H vector of the backbone This pattern suggests that a slow exchange process in the ls-to-ms range occurs at Tyr25, because such processes increase the value of the spectral density function in the lowfrequency range, i.e from zero to a few kilohertz, but have no influence at high frequencies, i.e in the megahertz range On the other hand, the significant J(0.87xH) value of Tyr25 indicates mobility Within the residue group with increased J(0), however, Tyr25 N–H and, to a lesser extent, His52 N–H appear to undergo some additional fast motions, as shown by higher J(0.87xH) values In general, flexibility is observed at the loop residues, but not in the tight turn For helix I and helix II, J(0) values show a quite regular distribution along the sequence if residues undergoing exchange are excluded (Fig 3) J(xN) and J(0.87xH) values are fairly constant along helix I [except for the higher value of J(0.87xH) for Leu16] and are more dispersed along helix II For the latter, this indicates segmental mobility being adopted with a less defined secondary sturucture, probably resulting from the lack of a complete hydrogen bond network [4] The dynamics of helix III can be divided into two different regions, with a border occuring at His52–Arg53 for all spectral density values The C-terminal segment of helix III (Arg53–Gln59) has lower J(0) values than the adjacent N-terminal moiety and the whole core of the TTF-1 HD, reflecting mobility related to poorly defined secondary structure [4] Conversely, at the N-terminal segment of helix III, higher J(0) values are inferred from analysis, consistent with the better defined and more stable secondary structure Overall, apart from the singularity at His52 that results from an exchange contribution due to slow aromatic ring motion, as previously described, J(0) values are seen to vary along helix III with some regularity within the two identified moieties, i.e a slight decrease along segment 47–53, followed by a slight increase in segment 53–55 The J(0) minimum is reached at Arg53, where the low-frequency motion profile shows similar characteristics as found at Arg58 for Gln59, the frayed extremity of the recognition helix The pattern described for J(0) is observed also for J(xN) along helix III, again with a minimum at Arg53 In a quite complementary fashion, J(0.87xH) reaches a maximum at Arg53, with a subsequent abrupt decrease at Tyr54 FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 441 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ă [in correspondence with the increases in J(0) and J(xN)] and then a progressive increase on moving towards the end of the recognition helix The whole picture outlines the peculiar dynamic profile of a hinge point at Arg53 that exhibits conspicuous minima of J(0) and J(xN) and a significant maximum of J(0.87xH), and that emerges not only within helix III, but also over a large portion of the protein, from Phe8 to Met56, including the loop and the tight turn At the same time, in the vicinity of the Arg53 hinge point, precisely at Glu50, a minimum of J(0.87xH) occurs, along with correspondingly high values of J(0) and J(xN), an indication of slow local motion consistent with the presence of a hydrogen bond network that restricts the excursion of the Glu50 backbone Other relevant details of the spectral density analysis are seen for Lys24 and Met37 amides, where increased values of J(0) are coupled to low J(xN) values Although significantly low values of J(xN) are considered to be evidence for fast motions, the corresponding J(0.87xH) of the same residues rather suggests more complex dynamics, i.e other than the dual low-frequency and high-frequency motional regime that appears to govern local dynamics elsewhere, e.g Leu26 Table lists the mean J(x) values together with the corresponding standard deviations for the different secondary structure elements of the TTF-1 HD It is readily seen that the 15N–1H vectors of helix I, helix II and the N-terminal segment of helix III not show major differences in the J(x) values Conversely, the C-terminal fragment of helix III has lower mean values for both J(0) and J(xN), and a significantly higher mean value for J(0.87xH), which further stresses the different dynamic behaviors of the N-terminal and C-terminal segments of the recognition helix Table Mean spectral density values (ns) and corresponding standard deviations (in parentheses) for the secondary structure elements of the TTF-1 HD at 286 K Structural unit J(0) Helix I (10–22) Helix II (28–38) Helix III (43–59) Helix III (43–52) Helix III (53–59) Loop (23–27) Tight turn (39–42) N-terminus (1–9) C-terminus (60–67) 3.78 3.84 3.63 3.83 3.38 3.79 3.69 2.83 2.62 a J(xN) (0.30) (0.37) (0.34) (0.26) (0.25) (0.12)a (0.11) (0.52) (0.30) 0.354 0.355 0.343 0.354 0.329 0.340 0.342 0.271 0.262 J(0.87xH) (0.008) (0.029) (0.020) (0.017) (0.014) (0.024) (0.010) (0.065) (0.052) 0.008 0.008 0.011 0.008 0.014 0.010 0.008 0.027 0.030 (0.001) (0.001) (0.005) (0.002) (0.006) (0.003) (0.001) (0.013) (0.006) Tyr25 was excluded to avoid a significant bias on the average from the slow exchange contribution (see text) 442 Global overall and generalized internal correlation times The roots of the third-order polynomial proposed by ` Lefevre [21] were calculated for both linear correlations of J(xN) and J(0.87xH) versus J(0) (see supplementary Fig S2) to evaluate sm and sgi From J(xN)– J(0) correlation, only two physically meaningful solutions were obtained, i.e sm = 9.85 ± 1.79 ns and J(0.87xH)–J(0) correlation sgi = 0.28 ± 0.11 ns yielded three roots, one for sm (9.84 ± 0.20 ns) and two for sgi (0.26 ± 0.03 ns and 0.55 ± 0.06 ns) (supplementary Doc S1 and Fig S2) Comparison of results from MF and RSDM The results for sm obtained by the MF and RSDM approaches are in fairly good agreement, especially if the comparison is drawn using the average value estimated from R2 ⁄ R1 ratios Therefore, the assumption of isotropic overall rotational diffusion for the TTF-1 HD proves to be convincingly appropriate The generalized order parameter values obtained from the MF approach are consistent with the results of RSDM Lower generalized order parameters are obtained for N-terminal and C-terminal residues, for the loop, and partially for helix III, pointing to largeamplitude motions Higher generalized order parameters are obtained for the structured regions as well as the tight turn, indicating restricted mobility, in agreement with the RSDM results Most of the effective internal correlation times obtained by the MF approach appear to be unreliable within the framework of the theory This could arise from the very wellknown limitations of MF formalism for the case of internal motions occurring on a timescale similar to that of the overall tumbling [27] In this case, such slow motions would superimpose faster internal motions, leading to a situation that would not match the regime supporting the assumption of MF formalism This is also assumed to be the reason why we were not able to fit our data using an extended MF formalism [26,27] Although anomalously high se values were often obtained, it is worth noting that MF calculations gave high S2 and correspondingly relatively low se values for Lys24, Glu30, Gln44 and Tyr54, indicative of restricted amplitude and fast-timescale motions that are consistent with the corresponding results from RSDM Also, the relatively decreased S2 and the corresponding relatively low se values (sub-nanoseconds) for Arg53, Arg58 and Gln59 suggest less restrictive and faster local motions that are consistent with the reduced spectral density results In the ls-to-ms timescale, only Tyr25 FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS D Gumral et al ă was found to have exchange contributions using MF analysis, whereas by RSDM, Lys21, Gln22, Ile38 and His52 were also identified Molecular dynamics (MD) simulation results Snapshots were taken at 500 ps intervals in order to obtain a statistical ensemble for the system studied The overall flexibility of the molecule was assessed by the average rmsd of the backbone atoms when the core region spanning residues Gln10–Arg58 was superimposed between all snapshot pairs From this analysis, the conformational freedom of the N-terminal and C-terminal regions was apparent, with average rmsd ˚ values up to 10 A The average rmsd values for the rest of the molecule spanning residues Gln10–Arg58 ˚ were mostly < 1.0 A The analysis of the correlation function of the N–H vectors was more informative, although less straightforward The short simulation time precludes a direct spectral density analysis In order to highlight local motions, the global rotational motion of the molecule must first be assessed This was done by superimposing the core of all snapshots, taken at 100 ps intervals, on the snapshot with the smaller average rmsd The correlation function C(i,m) was defined as the average of the position vector scalar r product ~NH tị ~NH t ỵ mDtị over the trajectory for r residue i The root mean square of the quantity [1 ) C(i,m)] was thus indicative of the deviation of the vector N–H of residue i from the global behaviour This procedure is solely motivated by the inadequate time sampling provided by a 10 ns MD simulation The largest deviations from global behavior are observed at the N-terminus and C-terminus, with a transition from disordered to more ordered vectors between Phe8 and Ser9, and between Gln59 and Arg58 Interestingly, this analysis highlights local motions at Gln10–Val13, Gln22–Ser27 and Met37– Leu40 and in the second part of helix III As could be expected, the analysis does not reproduce exactly the experimental findings, but it is consistent with them overall In particular, the long loop involving Gln22– Ser27 appears to be rather unconstrained, resulting in large conformational motions in its central part Similarly, the second part of helix III appears to be less restrained than the first part, starting from Tyr54 Arg53 appears to be more mobile than the preceding residues, but less free than the second part of the helix The pattern of hydrogen bonds is consistent with a regular a-helix throughout the simulation only for the first part of helix III Starting from Tyr54, the hydrogen bond with residue i-4 is not well conserved, and for Arg53 and Tyr54, hydrogen bonds with resi- Backbone dynamics of the rat TTF-1 homeodomain due i-3 are also observed, in good agreement with the helix tightening suggested by NMR Thus, the picture emerging from MD simulation is not as detailed as that provided by relaxation analysis, but it is consistent overall with the local motions observed by MF and ⁄ or RSDM analysis and with previous NMR structural findings Discussion The detailed description of the results obtained by the MF and RSDM approaches has highlighted a crucial limitation of the MF treatment When the motions of a protein in isotropic solution not match the regime of slow overall tumbling (nanoseconds) and fast local fluctuations (at most, hundreds of picoseconds), the MF-based fitting of the NMR relaxation data fails to retrieve a correct description of the dynamics As previously pointed out [27], there may be three major patterns of deviation from the basic MF assumption that can be hardly recognized when NMR relaxation is measured with a single magnetic field MF-based fitting does not apply properly when: (a) the overall rotation is anisotropic; (b) collective motions with correlation time longer than 1.5–2.0 ns are present; and (c) uniform conformational exchange occurs that may be masked by an overestimated sm For the experimental data of the TTF-1 HD presented here, it was concluded that only the two latter causes of deviation may contribute to the erroneous estimates obtained from MF analysis, although the possible uniform conformational exchange does not involve the whole molecule, but rather specific regions We could infer this conclusion from the simultaneous analysis of the data obtained using the RSDM approach The fitting obtained from the correlation plots among the different spectral densities ensures that the assumption of isotropic overall tumbling is correct within the experimental error This is consistent with previous evidence obtained for the vnd ⁄ NK-2 HD [28], which is very closely related to the TTF-1 HD, as well as with explicit anisotropy calculations that rule out anisotropic motion (supplementary Doc S1) The increase in the refined overall correlation time with respect to the average value obtained from relaxation rate ratios of single residues, within the MF context, most likely arose from inclusion in the dataset of the relaxation rates with slow exchange contributions (namely those from Lys21, Gln22, Ile38, and His52) The ensuing overestimated sm, in turn, obscured the detection of exchange contributions other than those of Tyr25 (which, in fact, was excluded from the dataset for refined sm calculation) Also, the sm value of FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 443 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ă 9.85 1.79 ns obtained from RSDM appears to be too large for a 68-residue polypeptide, and suggests the possibility of dimerization or higher-level association An estimate of the expected sm for a compact, globular protein, of the same molecular mass as the domain addressed here, gives values within 6.6 and 7.6 ns [29] Although aggregation into a stable dimer cannot be ruled out, in spite of the absence of structural evidence [4,11], the occurrence of association equilibria also cannot be excluded a priori, although the strong net charge of the molecule (+15) should prevent significant association Addressing this issue adequately, however, is beyond the scope of the current work, and should be done in detail elsewhere Besides the difficulty of demonstrating that the formation of a stable dimer or a labile, transient oligomer is capable of substantially affecting the internal dynamics of the monomers, so as to reject totally the conclusions of this study, it is worth considering the actual molecular dimensions to account for the large sm value In addition to the details that are discussed in supplementary Doc S1, one could mention that, as some 20 residues of the TTF-1 HD appear to be statistically disordered, the increment of the average hydrodynamic radius is well beyond 0.05 nm, which is expected to increase by 10% the overall sm [30] In fact, the Stokes–Einstein relationship gives a hydrodynamic radius of 1.98 nm for the TTF-1 HD under the conditions of this study, i.e very close to the mean radius of the NMR structure of the molecule (1.94 nm) [4] The conclusions inferred here may be much more intriguingly challenged if one wonders whether the dynamic properties of an isolated HD at 286 K can be extended to the whole TTF-1 molecule under physiological conditions The temperature increase at 310 K and the molecular size of the entire transcription factor should lead to an overall tumbling rate of 20–22 ns)1 Besides noting that the selected experimental conditions for characterizing the dynamics of the TTF-1 HD are not completely unrelated to the dynamic regime within the whole protein, it is clear that the local mobility trends that may influence HD function should still apply, and may possibly be elicited, under physiological conditions The most serious problem in MF interpretation of the TTF-1 HD data can be considered to be the coupling of restricted amplitudes and slow rates and, conversely, large amplitudes and faster rates, for the internal motions along most of the structured core of the molecule This picture is physically inconsistent, and follows from the failure to account for collective motions with correlation times > 1.5–2.0 ns [27] The possibility that the inconsistency is due to reliability 444 problems with measurements at a single field rather than inherent limits of the MF framework is in contrast to the results of interpretation of the same data obtained using the RSDM approach Despite the limitations, even with MF analysis, peculiar local fluctuation states were recognized at Lys24, Glu30, Gln44 and Tyr54, in agreement with the corresponding spectral density mapping interpretation In particular, it is instructive to consider the MF results obtained for Glu30 The arrangement of the helix II N-capping [4] seems to be paralleled by an increase in S2 and a decrease in se for Glu30 and, conversely, a decrease in S2 and an increase in se for the Arg31 15N–1H vector Thus, the result for individual se > smi obtained for Ser27, which is involved in N-capping with Arg31 N–H and Glu30 N–H is, at least qualitatively, justified, and suggests an interpretation based on the compensation between the amplitude and frequency of local fluctuations In other words, a wider motion amplitude is accompanied by a slower motion rate because of the increased mechanical inertia In the context of RSDM, the detailed analysis of the three spectral densities J(0), J(xN) and J(0.87xH) allowed us to obtain a rather complete description of the dynamics of the TTF-1 HD over a large range of timescales The current observations are in agreement with our previously published structural characterization of the TTF-1 HD [4] As we concluded before, the C-terminal segment of helix III, which is involved in the DNA recognition process, displays higher mobility than the preceding moiety, and Arg53 within the recognition helix appears to be a hinge point Additionally, slow conformational exchange contributions were observed for the His52 backbone, in a ls-to-ms timescale The high J(0) and J(xN) values obtained for the N-terminal moiety of helix III further stress its stability Within this first stretch of the recognition helix, Gln50 has a pivotal function High values of J(0) and J(xN) with a corresponding very low J(0.87xH) for the amide vector dynamics of this residue indicate local motions occurring essentially over the nanosecond timescale The lack of fast internal motions reflects the crucial role of Gln50, which behaves as mechanical point of support, needed for the hinging of the C-terminal part of helix III This relative rigidity of residue 50 is also relevant to biological function, and has been long recognized as one of the DNA recognition determinants of HD motifs [1,2] Slow motion contributions are seen to occur for Ile38 in the hydrophobic core or for residues close to this core (i.e Lys21 and Gln22, neighboring Phe20; Tyr25, neighboring Leu26), as well as for His52 (Fig 1), because of slow conformational exchange of FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS D Gumral et al ă an aromatic side chain from the same or a nearby residue Slow motion of aromatic side chains creates local field gradients at the neighboring residues, which may provide very efficient relaxation pathways, because of the well-known effects of ring currents on chemical shifts These contributions could be recognized as the collective slow motions that appear to occur along the helical backbone, as inferred from MF analysis failure The process seems particularly effective at the C-end of the helices, and could be regarded as helix–coil transition on a slow timescale [29] For the recognition helix, this behavior appears to correspond with the kink at Asn51–His52–Arg53; residues 51 and 53 are nearly invariant in all eukaryotic HDs, i.e are an early feature in HD evolution, and thus could represent a conserved determinant for the local dynamics [11] The resulting abrupt change of the recognition helix register introduced by the 51–53 kink, as confirmed by NMR evidence [4,11], should affect the amide bond vector dynamics of Tyr54, an important recognition determinant for the NK-2 HD subfamily [31], within the flexible joint between the N-terminal and C-terminal moieties of the recognition helix The implication for DNA binding that may be envisaged from the available conformational options within the recognition helix [2–4,7–9] is that the latter helix, firmly oriented within the helix–turn–helix motif, may undergo a transition, approximately in the middle of its extension, that alternates the local conformation between two limiting geometries involving either an extension or a break of the recognition helix This picture, first inferred for the TTF-1 HD from structural determination [4], proved consistent with the opposite stabilization pattern observed within the Asn51–His52–Arg53 segment through 1H–2H exchange measurements [11] The present relaxation study confirms our early interpretation [4] and provides support for our previous proposal The link between the nearly absolute invariance of Asn51 and Arg53 and the conformational dynamics of the recognition helix suggests that a double-bind is universally present in eukaryotic HDs, i.e an invariant termination signal for the first part of the recognition helix, and a likewise invariant resumption signal for the second part of the same helix at Asn51 and Arg53, respectively In behavioral science, doublebind designates two messages with conflicting meanings that are simultaneously submitted through two different communication channels The alternative arrangements and the conformational dynamics thereof are fully consistent with the hinging at Arg53 and can provide an important contribution to DNA recognition and binding These local collective transitions elicited at low temperature should still emerge Backbone dynamics of the rat TTF-1 homeodomain under physiological conditions, when the HD is part of a much larger transcription factor, and determine the extent of the conformational changes and, hence, the energetics of the interaction with DNA [2,3,8,11,32] Experimental procedures Sample preparation Uniformly 15N-labeled (U-15N) TTF-1 HD (68 residues including the segment 160–226 of the whole rat thyroid transcription factor, plus an extra methionyl residue at the N-terminus, numbered Met0) was obtained from overexpression in Escherichia coli strain BL21, by growth in a minimal medium containing 15NH4Cl as a source of nitrogen Expression and subsequent purification were performed as described previously [5,33] NMR samples were prepared by dissolving the lyophilized powder in H2O ⁄ D2O (95 : 5, v ⁄ v) and adjusting the pH (uncorrected pHmeter reading) to 4.3 by microadditions of m HCl The labeled protein concentration was about 0.8 mm NMR measurements The 2D NMR spectra were recorded at 286 ± 0.5 K and at 11.7 T on a Bruker (Karlsruhe, Germany) Avance500 spectrometer, operating at 500.13 MHz and 50.68 MHz for H and 15N, respectively The longitudinal (R1) and transverse (R2) 15N relaxation rate constants and steady-state {1H}–15N NOEs were measured from proton detection H–15N correlation spectra, according to schemes reported by Stone et al [34] All relevant chemical shift and relaxation rate data were deposited at BMRB (accession number 15521) Additional details can be found in supplementary Doc S1 15 N relaxation data analysis The longitudinal and transverse rate constants were calculated from peak heights of the 1H–15N correlation data series Under the typical conditions employed for protein NMR relaxation studies, peak heights have been proven to be more accurate than the corresponding volumes [35] To determine the R1 and R2 values, a three-parameter and two-parameter, respectively, nonlinear least-square fit of the equations I sị ẳ I1 I1 I0 ị expR1 sị 1ị I sị ẳ I0 expR2 sị 2ị and were applied, where s is the experimental relaxation delay, and I0 and I¥ are the initial and final steady-state intensities FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 445 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ă [35] Curve fitting was performed by means of the Marquardt–Levenburg algorithm implemented in the axum 5.0 package (MathSoft Inc., Cambridge, MA, USA) based on v2 optimization [35] The steady-state {1H}–15N NOE values were calculated from the height ratio of the peaks of 2D correlation spectra obtained with and without proton saturation, whereas the cross-relaxation rates, RNOE, were calculated according to the equation RNOE ẳ NOE 1ị cN R1 cH 3ị The details concerning the relaxation data analysis performed with the MF approach [14,15] and the RSDM approach [18–22] are given in supplementary Doc S1 Error estimations In order to establish the errors on individual peak height values, the reproducibility of the experimental R1 and R2 data was assessed by measurement duplication over a series of arbitrarily selected relaxation delays (at least three; see supplementary Doc S1) The average uncertainties obtained for R1 constants were 1.4% for resolved resonances and 1.0% for partially overlapping ones, whereas the corresponding quantities for R2 were 14% and 17% This difference reflects the inherent accuracy limit diversity of R1 and R2 estimations for a dilute sample at low temperature Analogously to the relaxation rates, the NOE data errors were also estimated by duplicated measurements and were analyzed only for a number of selected (resolved) resonances The estimation of the uncertainties affecting the dynamics parameters sm, S2, se and Rex was provided by covariance matrix analysis of the optimized model carried out by modelfree 4.1 software [23,35] and validated by comparison with the Monte Carlo simulation results obtained with the same package The uncertainties in spectral density functions were calculated according to standard error propagation equations using mathematica 5.2 software MD simulations MD simulations were performed starting from the deposited NMR structures (Protein Data Bank code: 1FTT), using the CHARMM forcefield [36] Overall, a 10 ns trajectory was simulated All the details are reported in supplementary Doc S1 Acknowledgements This work was financially supported by AIRC, MIUR (2006058958, RBNE03PX83) and EU (LSHM-CT2005-037525) The suggestions of Dr A Makek are acknowledged 446 References Gehring WJ 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Bellott M, Dunbrack RL Jr, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S et al (1998) All-atom empirical potential for molecular modeling and dynamics studies of proteins J Phys Chem B 102, 3586–3616 Supplementary material The following supplementary material is available online: Doc S1 Experimental details (NMR data acquisition and processing) FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS 447 Backbone dynamics of the rat TTF-1 homeodomain D Gumral et al ă Table S1 Assignment procedures and 1H and 15N chemical shift values Table S2 15N Relaxation rate, and {1H}–15N NOE values Table S3 Model-free smi, S2, se and Rex values Table S4 Spectral density function values Fig S1 Bar graph of R1, R2 and {1H}–15N NOE values along the sequence of the TTF1 HD at 11.7 T and 286 K 448 Fig S2 Plot of J(0.87xN)–J(0) correlation from 15N relaxation measurements of the TTF-1 HD This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 435–448 ª 2007 The Authors Journal compilation ª 2007 FEBS ... (0.04) (0 .19 ) (0.09) 19 83 2493 14 86 18 85 10 30 16 30 13 45 10 38 19 60 805 514 (406) (13 74) (780) ( 710 ) ( 613 ) (809) (740) (542) (10 47) (752) (277) 19 64 15 61 1008 15 96 905 12 97 11 42 468 14 03 276 206... S1 Bar graph of R1, R2 and {1H}? ?15 N NOE values along the sequence of the TTF1 HD at 11 .7 T and 286 K 448 Fig S2 Plot of J(0.87xN)–J(0) correlation from 15 N relaxation measurements of the TTF -1. .. dynamics of the HD recognition helix upon DNA binding [11 ] 436 In the following, we present a 15 N-NMR relaxation study of the rat TTF -1 HD to address the backbone dynamics in solution 15 N-NMR