Research

Interests

NMR-based Computational Structural Biology

Computation methods to study biomolecular systems, in particular by nuclear magnetic resonance (NMR), are the focus of our research. The relationship between structure, dynamics and function of biological macromolecules is of fundamental importance for understanding life at a molecular level, and a key element of rational drug design. The three-dimensional structure has a pivotal role, since its knowledge is essential to understand the physical, chemical, and biological properties of a protein. Until recently NMR protein structure determination was a laborious undertaking that occupied a trained spectroscopist for months or years for each new protein structure. This situation has changed by the introduction of automated, computational systems. We are extending NMR protein structure analysis to hitherto inaccessible systems, including proteins larger than 40 kDa, membrane proteins, and proteins studied directly inside living cells.

• Protein structure analysis
  

Fully automated NMR protein structure determination: Protein structures obtained by fully automated structure determination with the FLYA algorithm (blue) are virtually identical to the corresponding NMR structures determined by conventional methods (red). (a) ENTH domain At3g16270(9–135) from Arabidopsis thaliana. (b) Rhodanese homology domain At4g01050(175–295) from Arabidopsis thaliana. (c) Src homology domain 2 (SH2) from the human feline sarcoma oncogene Fes.

Three-dimensional structures of proteins in solution can be calculated on the basis of conformational restraints derived from NMR measurements. Our CYANA program package, based on simulated annealing by molecular dynamics simulation in torsion angle space and the automated assignment of NOE distance restraints, is one of the most widely used algorithms for this purpose. Automated methods for protein structure determination by NMR have increasingly gained acceptance and are now widely used for the automated assignment of distance restraints and the calculation of three-dimensional structures. Our FLYA algorithm for the fully automated NMR structure determination of proteins is suitable to substitute all manual spectra analysis and thus overcomes a major efficiency limitation of the NMR method for protein structure determination. Fully automated structure determination of proteins in solution (FLYA) yields, without human intervention, three-dimensional protein structures starting from a set of multidimensional NMR spectra. As in the classical manual approach, structures are determined by a set of experimental NOE distance restraints without reference to already existing structures or empirical molecular modeling information. In addition to the three-dimensional structure of the protein, FLYA yields backbone and side-chain chemical shift assignments, and cross peak assignments for all spectra.

[3] Koglin, A., Löhr, F., Bernhard, F., Rogov, V. V., Frueh, D. P., Strieter, E. R., Mofid, M. R., Güntert, P., Wagner, G., Walsh, C. T., Marahiel, M. A. & Dötsch, V. (2008). Structural basis for the selectivity of the external thioesterase of the surfactin synthetase. Nature 454, 907-911.

[4] Güntert, P. (2009). Automated structure determination from NMR spectra. Eur. Biophys. J. 38, 129-143.

[5] López-Méndez, B., & Güntert, P. (2006). Automated protein structure determination from NMR spectra. J. Am. Chem. Soc. 128, 13112–13122.

• Stereo-array isotope labeling
  

Fig. 2: Stereo-array isotope labeling (SAIL): The 20 standard amino acids are labeled such that each CH_n group carries at most a single NMR-visible ¹H nucleus, the others being replaced by NMR-invisible ²H. The remaining ¹H nuclei, shown as lights in the Figure, provide data that allows the NMR structure determination of proteins about twice as large as by conventional NMR approaches. The structure of the 42 kDa maltodextrin-binding protein MBP that is shown in the center of the Figure was solved in collaboration with the laboratory of Prof. Masatsune Kainosho at Tokyo Metropolitan University, Japan, using SAIL in conjunction with the structure calculation program CYANA.

NMR spectroscopy can determine the three-dimensional structure of proteins in solution. Nevertheless, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapped resonance lines and low signal-to-noise ratios. Stereo-array isotope labelling (SAIL) can overcome many of these problems by applying a complete stereo- and regiospecific pattern of stable isotopes, which is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL utilizes exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression such that in all methylene groups, one 1H is stereo-selectively replaced by ²H, in all single methyl groups, two ¹H are replaced by ²H, and in the prochiral methyl groups of Leu and Val, one methyl is stereo-selectively -¹²C(²H)₃ and the other is -¹³C¹H(²H)₂. In six-membered aromatic rings, the ¹²C-²H and ¹³C-¹H moieties alternate with each other. SAIL achieves a 4–7-fold increase in signal-to-noise, sharper resonance lines, and a 40–60% reduction in the number of signals without sacrificing essential information about the backbones and the side chains of all amino acid residue types. The resulting reduction of spectral overlap, the higher accuracy of the frequency determination, the complete stereospecific assignment, and the measurement of longer ¹H–¹H distances makes it possible to determine high-quality solution structures of proteins larger than 30 kDa.

Kainosho, M., Torizawa, T., Iwashita, Y., Terauchi, T., Ono, A. M. & Güntert, P. Optimal isotope labeling for NMR protein structure determinations. Nature 440, 52–57 (2006)

• Protein structure determination in living cells by in-cell NMR spectroscopy
  

Fig. 3: Protein structure determined in living cells: The first three-dimensional protein structure calculated exclusively on the basis of information obtained in living cells was solved by in-cell NMR for the putative heavy metal-binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in E. coli cells. A major hurdle for determining in-cell NMR structures is the limited lifetime of the cells inside the NMR sample tube. Standard NMR experiments usually require 1–2 days of data collection, which is an unacceptably long time for live cells. This time could be shortened to 2–3 hours by preparing a fresh sample for each experiment and by applying a nonlinear sampling scheme in combination with maximum entropy processing for the indirectly acquired dimensions.

Proteins in living cells work in an extremely crowded environment where they interact specifically with other proteins, nucleic acids, co-factors and ligands. Methods for the three-dimensional structure determination of purified proteins in single crystals or in solution are widely used and have made very valuable contributions to understanding many biological processes. However, replicating the cellular environment in vitro is difficult. In vivo observations of three-dimensional structures, dynamics and interactions of proteins are required for fully understanding the structural basis of their functions inside cells. Investigating proteins “at work” in a living environment at atomic resolution is thus a major goal of molecular biology. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and short life time of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Recently we presented the first three-dimensional protein structure calculated exclusively on the basis of information obtained in living cells. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

Sakakibara, D., Sasaki, A., Ikeya, T., Hamatsu, J., Hanashima, T., Mishima, M., Yoshimasu, M., Hayashi, N., Mikawa, T., Wälchli, M., Smith, B. O., Shirakawa, M., Güntert, P. & Ito, Y. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102-105 (2009)

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