Characterization of apo and partially saturated states of calerythrin, an EF-hand protein from S. erythraea: a molten globule when deprived of Ca(2+)

Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca(2+) and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca(2+)-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca(2+)-saturated states.     

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.     

Recognition of protein folds via dipolar couplings

Alignment of proteins in dilute liquid crystalline medium gives rise to residual dipolar couplings which provide orientational information of vectors connecting the interacting nuclei. Considering that proteins are mainly composed of regular secondary structures in a finite number of different mutual orientations, main chain dipolar couplings appear sufficient to reveal structural resemblance. Similarity between dipolar couplings measured from a protein and corresponding values computed from a known structure imply homologous structures. For dissimilar structures the agreement between experimental and calculated dipolar couplings remains poor. In this way protein folds can be readily recognized prior to a comprehensive structure determination. This approach has been demonstrated by showing the similarity in fold between the hitherto unknown structure of calerythrin and sarcoplasmic calcium-binding proteins from Nereis diversicolor and Branchiostoma lanceolatum with known crystal structures.     

A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H)-labeled proteins

Several HNCO-based three-dimensional experiments are described for the measurement of ¹³C(i–1)-¹³C(i–1), ¹⁵N(i)-¹³C(i–1), ¹⁵N(i)-¹³C(i), ¹⁵N(i)-¹³C(i–1), ¹H^N(i)-¹³C(i), ¹H^N(i)-¹³C(i–1), and ¹³C(i–1)-¹³C(i–1) scalar and dipolar couplings in ¹⁵N, ¹³C, (²H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.