SBNet - Research Reports 1998

PI: Forsén (LU)

Project: Protein structure and dynamics

The year 1998 witnessed the bearing of the first fruits of the forskarassistant position awarded in 1997. Two main projects are being pursued under this grant: [1] Structural and dynamical aspects of protein folding and [2] Structure, function and dynamics of a neuronal efficacy modulator.

[1] The first project deals with investigations of the mechanism by which the protein sequence information in one dimension is converted into the three-dimensional structure of a protein: i.e. the protein folding problem. The primary system being used for this study is the calcium-binding protein, calbindin D9k. This protein is well characterised both structurally and dynamically by both NMR spectroscopy and X-ray crystallography. It is a 75 amino-acid protein with a simple four-helix bundle structure and folds reversibly, making it an excellent model system for such studies. It is a member of a large family of closely related proteins, the co-called calmodulin superfamily or EF-hand superfamily, the latter name taken from the predominant folding motif of these proteins. In addition, a large number of mutants of calbindin D9k exist. During 1998 studies of the stability of the wild-type protein as well as a large number of mutants was completed as were complementary studies on structure and calcium binding properties. These were published back to back in Biochemistry (refs. 2 & 3). Further studies of this system are underway. A study of a related protein, S100B, was also completed under 1998. This involved modeling of the structure and calcium-induced conformational changes in S100B. Using a combination of modeling and high-resolution structural data on this and related proteins a prediction of the nature of the calcium-induced conformational changes in S100B were made. Following the publication of this prediction (ref. 1), two structures of S100B were published confirming this prediction.

[2] The second project involves the determination of the structure of a 190 amino acid protein identified as a regulator of neural efficacy in Drosophila and later also isolated from mammals (rat, human). The protein has partial homology with the recently characterised photoreceptor proteins, recoverin and neurocalcin. In addition to sequence similarity, they shares properties of N-terminal acylation and calcium-dependent activity. The protein has been produced in large quantities with both ¹⁵N and ¹³C labeling by Dr. Finns collaborators, Prof. Olaf Pongs and Dr. Alexander Hauenschild of Hamburg University. The complete resonance assignment was carried out using data from a battery of 3- and 4-dimensional triple resonance experiments on our 600 MHz Varian Unity+ NMR spectrometer. An article on these results is submitted. Structure calculation is also underway and we hope to complete the high resolution structure of the protein by mid-1999. Future directions for the project will include studies of the effects of calcium-binding on the structure and activity of the protein. Birthe Kragelund, a postdoc working on the project during the past two years, has taken a position at the Carlsberg Laboratory in Copenhagen but continues to be a collaborator on the project.

Collaborators

Lund University: University of Hamburg:

Bryan Finn Prof. Olaf Pongs

Karin Julenius (Graduate Student) Dr. Alexander Hauenschild

Carlsberg Laboratory:

Birthe Kragelund

Publications

(1) Groves, P., Finn, B. E., Kuznicki, J., Forsén, S. (1998) A model for target protein binding to calcium-activated S100 dimers. FEBS Lett. 421(3):175-179.

(2) Julenius, K., Thulin, E., Linse, S. and Finn, B. E. (1998) Hydrophobic core substitutions in calbindin D9k: Effects on stability and structure. Biochemistry. 37(25), 8915-8925.

(3) Kragelund, B. B., Jönsson, M., Bilfulco, G., Chazin, W. J., Nilsson, H., Finn, B. E. and Linse, S. (1998) Hydrophobic core substitutions in calbindin D9k: Effects on Ca2+ -binding and dissociation. Biochemistry. 37(25), 8926-8937.

(4) Muranyi, A., Finn, B. E., Gippert, G., Forsén, S., Stenflo, J. and Drakenberg, T. (1998) Solution structure of the N-terminal EGF-like Domain from Human Factor VII. Biochemistry. 37(30), 10605-10615.

(5) Finn, B. E. and Drakenberg, T. (1998) Calcium-binding proteins. Adv. Inorg. Chem.. 46, 441-494.

(6) Kragelund, B. B., Hauenschild, A., Carlström, G., Pongs, O. and Finn, B. E. (1998) ¹H, ¹³C and ¹⁵N resonance assignments, secondary structure and fold of Ca²⁺-frequenin, a synaptic efficacy modulator. Submitted.

Funding

Awarded a grant under the Cellulära signalsystem special programme of the SSF for the project: Structural characterisation of neuroproteins and their target complexes. The award is for two years (1999-2000).

Conferences

Organiser: Conference on NMR and Biomolecular Signaling: 17-19 September.

Oral presentations: SBNet Annual Conference, 8-11 May.

Poster presentations: Medicon Valley Academy Bio-conference: 8 September.

Conference on NMR and Biomolecular Signaling: 17-19 September.