Structural constraints for the Crh protein from solid-state NMR experiments
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We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples.
KeywordsCatabolite repression histidine-containing phosphocarrier protein (Crh) Distance constraints MAS 3D Protein structure Solid-state NMR spectroscopy
Solid state NMR
Magic angle spinning
Catabolite repression HPr-like protein
Proton driven spin diffusion
Solid state NMR (ssNMR) is rapidly developing to become complementary to solution NMR and X-ray crystallography as a method to study three-dimensional structure and dynamics in peptides and proteins. Because experiments can be conducted on insoluble and non-crystalline material, ssNMR is particularly well suited to investigate fibrous or membrane proteins at atomic resolution. Uniformly or multiply labeled protein variants enable the access to structural information regarding the complete polypeptide sequence using multidimensional ssNMR experiments. Even if structure determination has been shown to be possible using ssNMR techniques, no general protocol has been established today. Indeed, the strong one-bond couplings often hamper the measurement of distance constraints between carbon spins, and several approaches have been proposed to circumvent this problem. Extensive labeling strategies (Hong et al. 1999), removing one-bond couplings by partial labeling, have been used for the measurement of constraints and structure determination of two proteins so far (Castellani et al. 2002; Zech et al. 2005). Selective recoupling methods have been forwarded as another alternative (Böckmann et al. 2002). Indeed, it has been shown that the spin system dynamics remain sensitive to the distance of interest in chemical shift selective experiments and can be well reproduced within a quantum-mechanical multiple-spin analysis (Sonnenberg et al. 2004), making it possible to measure long-range constraints in uniformly labeled proteins. Selective transfer from carbonyl to side-chain carbons (Ladizhansky et al. 2004) or between nitrogen and carbon spins (Jaroniec et al. 2001) is another attractive approach for the measurement of long-range constraints. Recent work indicates that proton driven spin diffusion (Marulanda et al. 2005; Grommek et al. 2006) techniques are less sensitive to dipolar truncation. As an alternative to the measurement of carbon or nitrogen distances, a set of multidimensional NMR experiments for structure elucidation of proteins under MAS has been proposed, which relies on the measurement of proton distances (Heise et al. 2005). It uses proton homonuclear transfers, bracketed by 13C and/or 15N evolution times for the sake of spectral resolution. This scheme has been shown to be able to produce high resolution spectra connecting carbon or nitrogen spins through space (Lange et al. 2002, 2003), and we already applied this scheme successfully to the identification of the dimer interface of the Crh protein (Etzkorn et al. 2004). Most recent work shows that these heteronucleus-edited, proton relayed distance measurements can in principle be used for calculations of the 3D structure of solid proteins (Lange et al. 2005; Seidel et al. 2005). This approach is highly attractive, as it can be carried out on one uniformly labeled sample.
Here we explore the potential of this approach to collect local and long-range structural constraints for the larger and more complex 2 × 10.4 kDa Crh model protein in its dimeric, domain swapped form. This protein has proven to be an excellent model for solid state NMR methods developments (Carravetta et al. 2003; DePaëpe et al. 2003; Duma et al. 2003; Ernst et al. 2003; Juy et al. 2003; Lesage et al. 2003; Etzkorn et al. 2004; Giraud et al. 2004, 2005, 2006; Böckmann 2006; Böckmann et al. 2005; Lesage et al. 2006) as its solid state NMR chemical shifts and X-ray 3D structure (Böckmann et al. 2003) are available for reference. We show that a large number of proton-mediated constraints can be derived from CHHC and NHHC spectra at different mixing times. Information on side chain conformation and secondary structure, contained in intra-residue and sequential contacts, as well as long-range interactions constraining the 3D fold can be extracted from these spectra. Magnetization build-up curves confirm that the concept, established on small molecules, is valid also for proteins, contain detailed distance information and report on dynamic features of the molecule.
Materials and methods
Crh was overexpressed with a C-terminal LQ(6×His) extension as described previously (Galinier et al. 1997). Uniformly [13C,15N] labeled Crh was obtained by growing bacteria in Silantes growth media. The protein was purified on Ni-NTA agarose (QUIAGEN) columns followed by anion exchange chromatography on a Resource Q column (Penin et al. 2001). Crh-containing fractions were dialyzed against 20 mM NH4HCO3. We used 20 mg/ml protein concentrations as obtained after dialysis in sitting drops of 150 μl deposited on a siliconated crystallization glass plate (Hampton). The same volume of a 20% solution of PEG 6000 and 0.02% NaN3 in 20 mM NH4HCO3 was added to the drops, over a 2 M NaCl reservoir solution. The plates were left at 4°C until a precipitate appeared (1–2 weeks). Resulting microcrystals were directly centrifuged at 2000g into 4 mm Bruker CRAMPS rotors and rotor caps were sealed. The sample used in this study contained ca. 20 mg of protein.
NMR experiments were performed on Bruker AVANCE DSX 500 MHz wide bore and 700 MHz standard bore spectrometers, equipped with double (1H, 13C) and triple resonance (1H, 13C, 15N) Bruker MAS probes, at spinning speeds of 11 kHz. All experiments were carried out between −10 and −5°C probe temperature. Ramped cross-polarization (Metz et al. 1994; Hediger et al. 1995) was used in all experiments to transfer proton magnetization to the 13C or 15N spins. High power proton decoupling using the SPINAL-64 decoupling scheme (Fung et al. 2000) was applied during evolution and detection periods. The relaxation delay between scans was 2.5 s. 2D CHHC correlation spectra (Lange et al. 2002) with longitudinal proton mixing using spin diffusion were recorded using a first 1H → 13C 1 ms cross polarization (CP) period followed by two short CP steps of either 125 or 150 μs. Different sets of spectra were recorded, with (1H, 1H) mixing times ranging from 50 to 400 μs. Acquisition times were 20 ms in t2 and 7.5 or 7.9 ms in t1 respectively for experiments performed on the 500 and 700 MHz spectrometer, corresponding to a total acquisition time of 39 or 46 hours. The spectral width was 350 ppm in the acquisition dimension and 90 ppm in the indirect dimension, centered in the aliphatic region. The 1H r.f. field during SPINAL-64 decoupling and CP was set to 71 kHz and 56 kHz respectively. The carbon r.f. field during CP was 34 kHz. 2D NHHC correlation spectra with longitudinal proton mixing under spindiffusion were recorded using a first 1H → 15N 900 μs CP period. Short heteronuclear CP contact times tNH and tCH were 200 and 100 μs, respectively. Experiments were conducted with (1H, 1H) mixing times from 15 to 100 μs. Acquisition times were 30 ms in t2 and 10.5 ms in t1. The total acquisition time was either 35 or 45 hours. The spectral widths in the 13C and 15N dimensions were 350 ppm and 60 ppm, respectively. The 1H decoupling power was set to 71 kHz. R.f. field strengths for 15N and 13C during CP were 38 and 34 kHz, respectively.
Results and discussion
The recorded 2D CHHC and NHHC spectra show a large number of cross signals, including intra-residue, sequential, medium-range (2 ≤ ∣j–i∣ < 5) and long-range contacts (∣j–i∣ ≥ 5). A detailed analysis of these signals allows the extraction of distance constraints, to different degrees of precision, as we will show in the following.
As only very few carbon chemical shifts are unique when considering a resolution limit at best of ±0.25 ppm (corresponding to the experimental linewidth of 0.5–1 ppm), all 1H–1H contacts in the different NHHC and CHHC spectra presented in this work were identified according to the Crh dimer crystal structure (PDB code 1mu4 (Juy et al. 2003)) and the chemical-shift assignments for microcrystalline Crh (Böckmann et al. 2003). Correspondingly, isolated cross signals were assigned if their chemical shift was within ±0.25 ppm from a unique contact predicted from the crystal structure and associated to two protons with an interatomic distance smaller than 5 Å.
Intra-residue contacts yield information on secondary structure, as well as on side chain conformations. The angles ϕ and χ or combinations thereof directly relate to inter-proton distances as HN–Hα, HN–Hβ, Hα–Hβ, Hα–Hγ, Hβ–Hγ, which we denote here, in compliance with common practice in solution NMR, dNα dNβ, dαβ, dαγ, dβγ (Markley et al. 1998). To evaluate the correlation between cross peak intensities and distances in detail, magnetization build-up characteristics were measured. This analysis is possible in a more quantitative manner for heteronuclei with only one proton attached. This includes the amide protons, all Hα protons but Gly; Val, Thr, Ile Hβ, and Leu Hγ. Indeed, as NHHC and CHHC correlation experiments are based on rare-spin detection, there is no chemical shift signature for protons; as a consequence, these experiments do not allow the distinction between protons located on the same carbon or nitrogen. For a CH2 group for example, it is likely that both protons are affected in a different manner by the polarization transfer from an amide proton, and the detected signal will be the sum of two distinct transfers.
The same type of analysis can be conducted on intra-residue HN–Hβ correlations; the corresponding distance dNβ is distributed between 2 and 4 Å for the here considered Ile, Thr and Val residues showing a unique β-proton. We could identify isolated cross signals for Val8, Ile64, Thr59 and 62, where dNβ is around 2.5 Å, and for Thr30, with dNβ of 3.6 Å. The magnetization build-up curves are shown in Fig. 1d. The magnetization build-up for short distances is accordingly faster, enabling to distinguish short distances dNβ of 2–3 Å from longer distances (3–4 Å).
Considering intra-residue Hα–Hγ contacts of the leucine residues, with 1H–1H distances ranging from 2.9 to 3.3 Å, the isolated intra-residue Cα–Cγ signals of Leu10, Leu14 and Leu53 are absent from the spectrum shown in Fig. 3a, which is consistent with the observation of Hα–Hβ contacts mainly up to a distance of 2.7 Å in this spectrum, as discussed above.
In CHHC spectra, numerous sequential contacts between α-carbons can be identified. The corresponding sequential Hα–Hα distances dαα(i,i + 1) in the Crh protein range from 4.26 to 4.88 Å. As a consequence, the difference between the distance dαα(i,i + 1) for the different secondary elements is small. Moreover, although the sequential Hα–Hα contacts involve carbon atoms with only one proton attached, they correspond to 1H–1H distances longer than 4 Å. The observed 1H–1H contacts could therefore result from relayed transfer mechanisms involving other protons, as will be discussed further below. It is thus difficult to extract detailed information on the secondary structure from the presence or absence of sequential Hα–Hα cross signals at different (1H,1H) mixing times. As the longer distances correspond to proton pairs in α-helical secondary structure, only few sequential contacts in helices A and C can be detected even at relatively short (1H,1H) mixing times, whereas for helix B, no signals at all are observed even at longer mixing times (see Fig. 11 in the supporting information). The absence of cross signals for helix B is probably due to increased dynamics observed for this helix (see also below) (Favier et al. 2002).
Medium and long-range contacts
We have analyzed possible relay mechanism in a more detailed manner for unique CH···HC contacts identified in the spectrum recorded at 500 MHz with 200 μs (1H,1H) mixing time. Magnetization transfer between protons is mostly direct up to 3–3.5 Å, as previously observed by Baldus and coworkers (Lange et al. 2003). For longer distances, magnetization transfer is often relayed via a third spin. A succession of two short 1H–1H transfer steps, generally an intra-residue contact, followed by an inter-residue contact, may often result in the observation of long inter-residue contacts. Relayed transfers seem however not to introduce significant bias in distances; as can be seen from the buildup curves, the number of attached protons, as well as dynamic behavior (see below) play a preponderant role with respect to the degree of correlation between cross signal intensity and inter-nuclear distance.
Absence of predicted signals
We also analyzed predicted but not observed cross signals in the CHHC experiments. Missing cross signals in the distance range up to 4 Å mainly concern methyl groups (42%), which are difficult to polarize with short CP steps, but also labile sidechains of lysines and arginines (11%), or other Hγ (11%). Missing contacts corresponding to 1H–1H distances shorter than 4 Å, with the exception of methyl groups and arginine/lysine sidechains, are represented in Fig. 14 in the supporting information. Missing CH···HC contacts are mostly located in helix B, a result consistent with the identified missing NH···HC contacts. Several other missing signals are located in the hinge region, as well as in β1 and β4 strands and are also probably explained by dynamic features, these parts of the protein being involved in the dimer interface. As in solution NMR, the ultimate proof that dynamic behaviour is at the origin of missing signals can only be brought (else than by backcalculation of expected signals from an existing structure) by dynamic measurements, which can confirm that regions with missing cross peaks indeed show increased dynamics. Several of such measurements have been proposed for solid proteins over the past years (Giraud et al. 2004, 2005; Hologne et al. 2005, 2006; Giraud et al. 2006, 2007; Reif et al. 2006; Chevelkov et al. 2007a, b; Xue et al. 2007). Measurements at low temperatures could also be used to establish if dynamics is at the origin of missing signals; however, line broadening hampers measurements as soon as freezing occurs.
We have shown here that proton mediated rare-spin detected experiments yield distance information including intra-residue, short, medium and long-range contacts, and are thus valuable for constraining 3D protein structures. In contrast to carbon correlation spectra, every single cross signal present in the spectra potentially contains distance information, as has already been shown for proton contacts in solution NMR. Cross signals connecting carbon or nitrogen spins with only one proton attached reflect very well the distance encompassed. This is especially true for intra-residue and sequential connections, and, to a slightly lesser extent, also for medium- and long-range contacts. In general, build-up curves for local contacts correlate better to the distances than long-range constraints. The dipolar couplings at the origin of the cross signals depend on the distance as well as the orientation of the spin-pair. If molecular motion occurs during the mixing time, it may provoke a change in orientation leading to partial averaging of the dipolar coupling, resulting in signal attenuation. This is typically the case for cross signals involving lysine and arginine side chains. Larger scale flexibility between secondary structure elements is often involved in function, like molecular recognition, catalysis site function or conformational change in the global folding of the protein, and may lead to the absence of predicted cross signals, as illustrated above. Even if dynamic behavior might attenuate magnetization transfer, we did not observe, in this microcrystalline Crh protein sample, regions completely devoid of corresponding cross signals. This situation seems similar to solution NMR, where less contacts are observed for mobile regions of the protein, without however compromising structural information for well-folded regions of the protein. Another factor, possibly limiting the direct extraction of distance classes from the magnetization build-up curves, is that medium- and long-range contacts may be more subject to relay mechanisms than intra-residue or sequential contacts, as already observed in solution NMR. Consequently, the model of an isolated spin pair is not valid any more, preventing a detailed comparison of the magnitude of the observed cross signals. Even if present, relay mechanisms are still less important for proton connectivities than in carbon correlation experiments, where magnetization transfer between directly bonded carbon spins is complete within a short time compared to the mixing times required for long-range magnetization transfers. Long-range information up to 5 Å can easily be detected in the here presented NHHC and CHHC spectra. It is difficult to evaluate if contacts involving longer distances are present as well, as spectral crowding and assignment ambiguities make an analysis difficult. Our analysis in Fig. 9 shows however that no clear limit is indicated by the cross signals which could be identified, and potentially cross signals corresponding to longer distances might be present. The accuracy of these constraints could however be limited by relayed magnetization transfer mechanisms, and might not add essential information for high-resolution structure determination. Sensitivity remains an issue in proton mediated experiments, but the necessary amount of sample and acquisition time should decrease with the use of higher fields.
We identified a large number of constraints in the CHHC and NHHC spectra for resolved signals, using the information available from the crystal structure and a maximum 1H–1H transfer range of 5 Å. Without structural information, unambiguous signal assignment is impossible for the high number of spins in the Crh protein combined with linewidths of 0.5–1.0 ppm, even if three-dimensional experiments would be used. Structure calculation for this size of proteins from proton-mediated (and also from carbon or nitrogen correlation) experiments will probably mainly be carried out using protocols including automated iterative assignment procedures to resolve ambiguities in the distance restraints, as implemented in software routines such as ARIA (Nilges 1995), SOLARIA (Fossi et al. 2005) or CYANA (Güntert et al. 1991). We will report on this for the Crh protein in a forthcoming paper.
Electronic supplementary material
A graph presenting Hα–Hα distances identified in CHHC spectra, assignments of the NHHC spectrum, additional magnetization build-up curves as well as an analysis of absent signals for the CHHC experiments are given.
This work has been supported by the Centre National de la Recherche Scientifique (PICS no. 2424), the ANR (JC05_44957) and the French Ministry (ACI Biologie cellulaire moléculaire et structurale). CG acknowledges a Claudie-Heigneré post-doctoral grant.
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