Solvent-induced differentiation of protein backbone hydrogen bonds in calmodulin

Abstract

In apo and holoCaM, almost half of the hydrogen bonds (H-bonds) at the protein backbone expected from the corresponding NMR or X-ray structures were not detected by h3JNC' couplings. The paucity of the h3JNC' couplings was considered in terms of dynamic features of these structures. We examined a set of seven proteins and found that protein-backbone H-bonds form two groups according to the h3JNC' couplings measured in solution. H-bonds that have h3JNC' couplings above the threshold of 0.2 Hz show distance/angle correlation among the H-bond geometrical parameters, and appear to be supported by the backbone dynamics in solution. The other H-bonds have no such correlation; they populate the water-exposed and flexible regions of proteins, including many of the CaM helices. The observed differentiation in a dynamical behavior of backbone H-bonds in apo and holoCaM appears to be related to protein functions.

Figure 1.

Sequential NOE distances of amide protons in apoCaM (Zhang et al. 1995a) (top, backbone ^h3 J _NC′ couplings [second from top]) and relative sensitivity of J-HNCO experiment (third from top). (Central insert) The secondary structure of apoCaM; (rectangular boxes) helices; (arrows) the short β-sheet contacts. Detected β-sheet contacts are labeled with the corresponding residue numbers. (Three bottom inserts) Magnitudes of the ^h3 J _NC′ ^calc couplings that we calculated for three of the reported structures (Kuboniwa et al. 1995; Zhang et al. 1995a) (in the 1QX5 structure [Schumacher et al. 2004], there are several intermolecular H-bonds [light-gray bars]).

Figure 2.

Strips from 3D HNCO-J spectrum displaying NC′ correlation peaks in the H-bond chain 107/108 → 104/105 → 101/102 → 102/134. The spectrum, recorded at the J-coupling evolution time of 1/4J = 32 msec, is the sum of two 3D HNCO-J spectra each collected for 4 d on a Bruker 600 MHz spectrometer equipped with a cryo-probe.

Figure 3.

Relative sensitivity of J-HNCO experiment (top) and backbone ^h3 J _NC′ couplings (second from top). (Central insert) The secondary structure of holoCaM; (rectangular boxes) helices; (arrows) the short β-sheet contacts. Detected β-sheet contacts are labeled with the corresponding residue numbers. (Bottom inserts) The magnitudes of the ^h3 J _NC′ ^calc couplings that we calculated for two of the reported structures (Wilson and Brunger 2000; Chou et al. 2001).

Figure 4.

Hydrophobic contact of helices E and H in holoCaM. (Dashed lines) The H-bonds detected by ^h3 J _NC′ couplings (magenta-colored region of the helices). (Top part) The peptide-bond ¹ J _NC′ couplings in the E-helix of apo and holoCAM.

Figure 5.

Angle–distance correlation of H-bonds from seven proteins (crambin, ubiquitin, protein G, parvalbumin, IFABP, and apo- and holo-calmodulin) divided into two subsets according to the magnitude of the H-bond couplings (|^h3 J _NC′| > 0.2 Hz, top; |^h3 J _NC′| < 0.1 Hz, bottom). The distance correlation for the donor angle, Δα_H (○), is matched with the correlation for the acceptor angle, Δα_O (□), upon scaling the latter by 0.72. (The full curved line) The fifth-power distance dependence (0.8d _OH ⁵); (dashed lines) ∼90% containment boundaries. The peptide groups involved in the coordination to Ca²⁺ were excluded from the analysis.

Figure 6.

Experimental RDCs of peptide groups (Δ¹ J _HN, Δ¹ J _NC/0.12, and Δ¹ J _CCa/0.20) in α-helices of the holoCaM C terminus, in comparison with the calculated values for the corresponding crystal and solution structures. The crystal structure is represented by X-ray high-resolution structure 1exr-model-A (Wilson and Brunger 2000), while the solution structure is modeled from the X-ray structure to satisfy RDCs. The high-J and low-J data refer to the size of proton-donating H-bonds of the peptide groups. (Lower insert) The X-ray and the modeled solution structure of helix E. (Small arrows) Connection between peptide-group orientations and RDC fit for the peptide group in position 91/92. The hydrophobic anchoring residue is indicated.

Figure 7.

Comparison of the high-J subset H-bonds in apoCaM (top) and holoCaM (bottom). The population of the helices with the detected H-bonds is represented by the shades of the rectangular boxes: (black) >70%; (gray) ∼50%; (empty) <30%. Detected β-sheet contacts are labeled with the corresponding residue numbers.