Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Abstract

All the backbone 1H and 15N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by 15N-edited two-dimensional NMR experiments on selectively 15N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly 15N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or C alpha protons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C alpha protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six beta stands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the beta-sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.

NMR Characterization of Full-length Farnesylated and Non-farnesylated H-Ras and its Implications for Raf Activation

“…13,[22][23][24][25][26] The binding of GTP to Ras causes a conformational change in two regions, commonly referred to as switch I (residues [32][33][34][35][36][37][38][39][40] and switch II (residues 58-75). [27][28][29] The GTP-dependent conformation of switch I is critical for high affinity interaction between Ras and the Raf-RBD.…”

ATR-FTIR Spectroscopy of Membrane Bound Ras Protein

“…This was justifying the applicability of the parameters for the forthcoming measurements. Commonly, the NMR studies on the Ras protein revealed higher unstructured contents which were counterbalanced by roughly equal amounts of diminishes in other structural elements, while the overall structures were mainly consistent with the crystal structures [123,124]. This information was pointing at the contribution of the protein dynamics enhancement in solution.…”

“…This was making part of the β-sheet structure flexible and the information was contrasting to the rigidity in crystalline states [126]. In relation, it was suggested [123] that the salt bridge between D33 of one Ras protein molecule and R135 of another symmetryrelated molecule in the crystalline state [126] was stabilizing the loop L2 conformation and the end of antiparallel β-strand β2. Besides, the residue N26 which was found to be essential for the signal transducing activity of Ras [136], was exhibiting a flexible nature in solution [123].…”

“…In relation, it was suggested [123] that the salt bridge between D33 of one Ras protein molecule and R135 of another symmetryrelated molecule in the crystalline state [126] was stabilizing the loop L2 conformation and the end of antiparallel β-strand β2. Besides, the residue N26 which was found to be essential for the signal transducing activity of Ras [136], was exhibiting a flexible nature in solution [123]. This was also suggested to have the potential to be ascribed to the intermolecular contact of loop L2 in the crystal structure [126].…”

“…possessing the residue E49 was reported to be flexible in solution [123], in contrast to the rigidity in crystalline states [126]. It was also stated [42] that the K170 and R169 had the potential to form H-bonds with the charged residues of the lipid phosphate head groups.…”