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Observation of Ls Time-Scale Protein Dynamics in the Presence of Ln Ions

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Observation of Ls Time-Scale Protein Dynamics in the Presence of Ln Ions J Biomol NMR (2007) 37:79–95 DOI 10.1007/s10858-006-9105-y ARTICLE Observation of ls time-scale protein dynamics in the presence of Ln3+ ions: application to the N-terminal domain of cardiac troponin C Christian Eichmu¨ ller Æ Nikolai R. Skrynnikov Received: 20 September 2006 / Accepted: 2 October 2006 / Published online: 19 December 2006 Ó Springer Science+Business Media B.V. 2006 Abstract The microsecond time-scale motions in the environment of the reporting spin does not change. This N-terminal domain of cardiac troponin C (NcTnC) happens, for example, when the motion involves a ‘rigid’ loaded with lanthanide ions have been investigated by structural unit such as individual a-helix. Even more means of a 1HN off-resonance spin-lock experiment. significantly, the dispersions based on pseudocontact The observed relaxation dispersion effects strongly shifts offer better chances for structural characterization increase along the series of NcTnC samples containing of the dynamic species. This method can be generalized La3+,Ce3+, and Pr3+ ions. This rise in dispersion effects for a large class of applications via the use of specially is due to modulation of long-range pseudocontact shifts designed lanthanide-binding tags. by ls time-scale dynamics. Specifically, the motion in the coordination sphere of the lanthanide ion (i.e. in the Keywords Cardiac troponin C Á Spin-lock relaxation NcTnC EF-hand motif) causes modulation of the dispersion experiment Á ls–ms protein dynamics Á paramagnetic susceptibility tensor which, in turn, causes Lanthanide Á Pseudocontact shift Á Calmodulin modulation of pseudocontact shifts. It is also probable that opening/closing dynamics, previously identified in Ca2+–NcTnC, contributes to some of the observed Introduction dispersions. On the other hand, it is unlikely that monomer–dimer exchange in the solution of NcTnC is Protein folding, protein–protein and protein–ligand directly responsible for the dispersion effects. Finally, interactions are all essentially dynamic processes that on–off exchange of the lanthanide ion does not seem to are often predicated on complex internal motions play any significant role. The amplification of dispersion which occur on a ls–ms time scale. For studies of ls– effects by Ln3+ ions is a potentially useful tool for studies ms dynamics a number of NMR experiments have of ls–ms motions in proteins. This approach makes it been developed over the course of years (Palmer 2004; possible to observe the dispersions even when the local McDermott 2004). Notably, these experiments are uniquely suited for characterization of dynamic equi- Electronic supplementary material Supplementary material is libria involving ‘excited states’, i.e. transient low-pop- available in the online version of this article at http://www.dx. ulation species that often play a crucial role in the doi.org/10.1007/s10858-006-9105-y and is accessible for biological function of proteins (Mulder et al. 2001). authorized users. The data from NMR experiments are usually con- sistent with a simple two-state exchange model, where C. Eichmu¨ ller Á N. R. Skrynnikov (&) Department of Chemistry, Purdue University, 560 Oval each of the states is characterized by distinct chemical Drive, West Lafayette, IN 47907-2084, USA shifts. The chemical shift difference between the two e-mail: [email protected] states, Dx, is central to NMR analyses of exchanging systems. Under fast exchange conditions, Dx manifests Present Address: C. Eichmu¨ ller itself through line broadening and can be accessed by Sandoz GmbH, Biochemiestraße 10, A-6250 Kundl, Austria means of relaxation dispersion experiments (Bloom 123 80 J Biomol NMR (2007) 37:79–95 et al. 1965; Deverell et al. 1970). In this work we study Skrynnikov 2005) to record relaxation dispersion pro- the dynamic system where the difference in chemical files. In the presence of paramagnetic lanthanides the shifts is augmented by the difference in pseudocontact dispersion amplitudes were strongly increased. We shifts, induced by Ln3+ ions. attribute this effect primarily to ls time-scale dynamics The proposed method is demonstrated for the in the lanthanide binding site. This dynamics leads to N-terminal domain of cardiac troponin C. Troponin C modulation of paramagnetic susceptibility tensor which, (TnC) is a dumbbell-shaped protein which, together in turn, causes modulation of long-range pseudocon- with troponin I (TnI) and T (TnT), forms the troponin tact shifts and thus gives rise to line broadening and assembly. The C-terminal lobe of TnC possesses two dispersion effects. high-affinity metal-binding sites that are permanently We have also analyzed other possible forms of occupied under physiological conditions. This domain dynamics contributing to Ln3+-induced dispersions. The plays a structural role as it anchors the N-terminal helix data suggest that opening/closing motion such as iden- of TnI. In contrast, the N-terminal domain (NcTnC) tified in Ca2+-NcTnC may well contribute to the ob- serves as a Ca2+-regulated molecular switch. Only one served dispersions. On the other hand, it is unlikely that of the two EF-hand motifs in NcTnC is capable of transient dimerization of Ln3+-NcTnC is directly binding calcium—the other site is defunct due to responsible for the dispersion effects, although it may alteration of the two ligand residues. Upon loading a influence other forms of ls motion. On-off exchange of single Ca2+ ion, NcTnC switches to the dynamic state Ln3+ ion does not seem to be a significant factor under that is primed for binding (Pa¨a¨kko¨ nen et al. 1998; the conditions of our experiments, and there is no reason McKay et al. 2000; Eichmu¨ ller and Skrynnikov 2005) to suggest a presence of secondary Ln3+-binding sites. and, consequently, locks onto a short amphiphilic helix In conclusion, we discuss the prospects for using Ln3+ III from TnI (Li et al. 1999). As a result, a large por- ions as probes of native protein dynamics in the ls–ms tion of TnI is displaced, including the two so-called range. Remarkably, long-range pseudocontact shifts inhibitory regions located up- and down-stream from can generate large dispersions where chemical shifts fail the helix III (Farah et al. 1994; Takeda et al. 1997; Luo because of their local nature. Even more significantly, et al. 2000). These inhibitory regions are lifted off of the values of Dx associated with pseudocontact shifts their binding sites on the surface of actin, thereby can be readily converted into structural parameters. bringing about changes in the actin-tropomyosin This paves the way for the structural characterization of assembly and clearing the way for actin–myosin bind- the exchanging species, which is especially valuable in ing. The result is a contraction of a striated muscle the case of the elusive ‘excited states’. However, this (Kobayashi and Solaro 2005). approach is feasible only if the Ln3+ ion is tightly bound Like other calcium-binding proteins (Campbell in a well-defined static environment and does not et al. 1975; Lee and Sykes 1983; Bentrop et al. 1997), interfere with native protein dynamics. From this per- NcTnC binds Ln3+ ions in its calcium-binding site spective, lanthanide-binding tags present an attractive (Wang et al. 1981; Gay et al. 2004). Lanthanide ions and sufficiently general option. have a well-established record as useful structural probes in NMR (Lee and Sykes 1983; Dwek et al. 1971), X-ray crystallography (Weis et al. 1991; Burling Observation and analysis of Ln3+-induced dispersions et al. 1996), and luminescence spectroscopy of proteins (Rhee et al. 1981). Particularly, in NMR studies con- The affinity of Ca2+ to NcTnC is ca. 3 lMat30°C(Li tact and pseudocontact shifts, various relaxation et al. 1997; Hazard et al. 1998) and probably somewhat enhancement parameters, and residual dipolar cou- tighter at lower temperatures (Li et al. 2002). The plings due to Ln3+-induced alignment were all used for titration of Ca2+, therefore, proceeds under interme- protein structure determination (Bertini et al. 2001a). diate-to-fast exchange conditions with each resonance In addition, Ln3+-for-Ca2+ substitution helps to eluci- moving in a continuous fashion. In contrast, the La3+, date the structural mechanism of calcium regulation Ce3+ and Pr3+ titration is characterized by a slow (Mustafi et al. 2004; Dudev et al. 2005). In this work exchange regime, with apo and holo forms giving rise we extend the scope of Ln3+ applications by demon- to distinct sets of peaks. In this situation, one cannot strating that lanthanides can be used to ‘highlight’ the rely on a lanthanide titration to obtain complete effects of ls–ms time-scale motion in proteins. spectral assignment of Ln3+-NcTnC. Instead, a set of We have prepared the samples NcTnC with La3+, triple-resonance experiments was executed for each of Ce3+, and Pr3+ ions and used our recent proton off- the three samples and the backbone assignment was resonance spin lock experiment (Eichmu¨ ller and thus obtained. The 1H,15N-HSQC spectra of the 123 J Biomol NMR (2007) 37:79–95 81 NcTnC samples loaded with different ions are should bear in mind, however, that (i)Dy3+ generates presented in Fig. 1. much larger shifts than Ce3+ or Pr3+,(ii) in contrast to The analysis of the lineshapes in the course of the NcTnC, parvalbumin has very little internal motion lanthanide titrations established that the Ce3+ off-rates (Baldellon et al. 1998), and, on a related note, (iii) the at 30°C are in the range of 15–25 s–1, while Pr3+ off- structural model of parvalbumin has somewhat higher rates at 10°C are 5–10 s–1. Assuming that lanthanides precision. Of note, Ca2+-NcTnC also produced a rela- bind to NcTnC in diffusion-controlled manner, just like tively poor fit in the study employing 15N-1H residual calcium (Hazard et al.
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