Secondary structure of lipidated Ras bound to a lipid bilayer

Abstract

Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post‐translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance‐FTIR studies of lipid‐modified membrane‐bound N‐Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N‐Ras was prepared by linking a farnesylated and hexadecylated N‐Ras lipopeptide to a truncated N‐Ras protein (residues 1–181). It was then bound to a 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three‐dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane‐bound lipidated Ras, and provided a reliable decomposition for the membrane‐bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane‐bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G‐domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane‐bound Ras protein.

Revealing conformational substates of lipidated N-Ras protein by pressure modulation

“…S1). As expected, no major secondary structural changes or even unfolding are observed in the protein upon membrane insertion, in agreement with previous reports (23,33). Significant conformational changes are identified in the second derivative spectra for N-Ras (GDP) and N-Ras (GTP) inserted into the membrane upon pressurization, however (Figs.…”

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA fromAnabaenasp. PCC 7120

“…The signal-to-noise ratio of the spectra was better than 10.000:1. Baselines were subtracted, and contributions of side chains removed as described (16,17). Only in those cases where there were no distortions in the spectra, the resulting difference spectra were used for analysis.…”

Membrane extraction of Rab proteins by GDP dissociation inhibitor characterized using attenuated total reflection infrared spectroscopy

“…The spreading was complete after 10 min. The vesicles ruptured quickly upon adsorbing to the germanium crystal because no overshoot kinetics was observed, which is typical for the accumulation of intact vesicles at the surface (19). This strong interaction between vesicles and germanium is probably due to the negative charge introduced by DOPS because neutral vesicles show slower spreading kinetics with an overshoot (19).…”

“…Various techniques exist for the immobilization of proteins on the lipid bilayer, the most natural for small GTPases being via lipid anchors attached to the protein. The availability of lipidated Ras GTPase allowed investigation of the secondary structure and orientation of membrane-bound Ras with ATR-FTIR spectroscopy, and recent advances allow recording of difference spectra of protein activity, such as GTP hydrolysis (15,19,20).…”

“…Model membranes can be formed by vesicle spreading on the internal reflection element, such as a germanium crystal (18,19). Various techniques exist for the immobilization of proteins on the lipid bilayer, the most natural for small GTPases being via lipid anchors attached to the protein.…”