Structure determination
In the solid state
Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins
Petra Rovó, Kristof Grohe, Karin Giller, Stefan Becker, Rasmus Linser
Solid‐state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic‐resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long‐distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton–proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin‐labeled 1 mg protein samples using fast magic angle spinning.
In the solution state
Cooperativity network of Trp-cage miniproteins: Probing salt-bridgess
Petra Rovó, Viktor Farkas, Orsolya Hegyi, Orsolya Csikós, Gábor Tóth and András Perczel
Trp‐cage miniprotein was used to investigate the role of a salt‐bridge (Asp9–Arg16) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side‐chains are also part of a dense interaction network composed of electrostatic, H‐bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt‐bridge with those of the salt‐bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp‐cage miniproteins is more complex than a simple two‐state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt‐bridge stabilization; in the miniprotein variants equipped with salt‐bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt‐bridge deleted mutants. In this molecular framework Arg16 stabilizes more than Asp9 does, because of its higher degree of 3D‐fold cooperation. In conclusion, the salt‐bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network.