Prof. Dr. Harald Schwalbe

NMR and biophysical characterization of structure and dynamics of m-proteins and their complexes

Goethe University Frankfurt am Main (Germany), Center for Biomolecular Magnetic Resonance (BMRZ), Institute for Organic Chemistry and Chemical Biology

Nuclear magnetic resonance (NMR) spectroscopy represents a powerful technique to determine the structure and dynamics of proteins in solution in isolation but also in complexes with diverse cellular components. In particular, NMR provides an unbiased readout of the folding state of small proteins including folded, partially folded as well as unstructured (random coil) states, the almost likely states for these small proteins.

Within the DFG Special Priority Program (SPP) 2002, the group of Harald Schwalbe collaborates with a large number of other groups. We have already investigated 25 different small proteins1, 2. We established a work protocol for fast secondary structure screening. Typically, for polypeptide chains with less than 30 amino acids, we utilize solid phase peptide synthesis (SPPS), while target sequences with more than 30 amino acids are expressed in E. coli as SUMO-fusion proteins. The purification is performed either by reversed-phase HPLC or by tandem Ni-NTA affinity chromatography and SUMO cleavage. The identification and the purity of produced small proteins and peptides are confirmed by SDS-PAGE analysis and mass spectroscopy (MALDI).

Circular dichroism (CD) and 1D 1H-NMR spectroscopy provide information about the secondary structure. Moreover, the 2D 1H15N-HSQC-NMR experiment exposes the structural characteristics of the biomolecule in more detail. The dispersion of the detected backbone amide signals and their line widths are markers for the three possible conformational states: structured, unfolded/unstructured or partially structured and undergoing ms-intermediate exchange. Chemical shift analysis by TALOS allows a detailed and reliable site-specific secondary structure prediction. Such analysis is based on the backbone assignment of the small proteins.

Figure 1: left panel: 60mer P14 containing peptide from Haloferax volcani; lmiddle panel: (SP26_1_SW) 23mer P12 containing peptide from Methanosarcina mazei; right panel: 31mer P14 containing peptide from Sinorhizobium fredii. A: CD-spectra of peptide in different phosphate buffer pH 7. B: matrix-assisted laser desorption/Ionization (MALDI) mass analysis of the purified peptide. C: 1D 1H-NMR-spectra with amid proton region enlarged. D: 2D 15N-NMR-spectrum 600 MHz, 298 K, resonance assignment as indicated. E: TALOS secondary structure prediction of residues which are classified as “good”.

Figure 2: 60mer P17 containing peptide from Haloferax volcanii (collaboration with A. Marchfelder); left panel: 2D 1H,15N-HSQC NMR-spectra measured at 600 MHz and 298 K with the backbone amide assignment indicated; right panel: Representative structure of the dimer and a bundle of 20 conformers.

For the small protein P17 from Haloferax volcanii (collaboration with A. Marchfelder), we could show that it adopts a persistent structure. In this case, we record standard triple resonance 3D NMR experiments for 1H, 13C, 15N backbone and side chain assignment and obtain NOE distance restraints for structure calculations. The initial structure of the protein suggested an α-helix and four β-sheet regions with a monomeric structure. Further investigation of the dynamical properties by heteronuclear 15N relaxation experiments ({1H}-15N hetNOE, 15N-T1 and -T2) and comparison of the experimental rotational correlation time (τc) with the values obtained by using HydroNMR show that protein actually forms a dimer. This is supported by the additional protected hydrogen bonds observed in the 3rd beta sheet and confirmed by the better quality of the calculated dimeric structure.

Team: Nina Kubatova, Laura Remmel, Kerstin Witt (PhD funded); Henry Jonker, Krishna Saxena, Kerstin Witt, Harald Schwalbe

  1. J. Grote, D. Krysciak, K. Petersen, S. Güllert, C. Schmeisser, K. U. Förstner, H. B. Krishnan, H. Schwalbe, N. Kubatova, and W. R. Streit, “The Absence of the N-acyl-homoserine-lactone Autoinducer Synthase Genes traI and ngrI Increases the Copy Number of the Symbiotic Plasmid in Sinorhizobium fredii NGR234,” Front. Microbiol., vol. 7, no. November, p. 1858, 2016.
  2. J. Hahn, S. Thalmann, A. Migur, R. Freiherr von Boeselager, N. Kubatova, E. Kubareva, H. Schwalbe, and E. Evguenieva-Hackenberg, “Conserved small mRNA with an unique, extended Shine-Dalgarno sequence,” RNA Biol., vol. 0, no. 0, pp. 00–00, 2016.