NMR STUDIES ON STRUCTURE AND DYNAMICS OF TWO-DOMAIN PROTEINS

Abstract: The general objective of the present thesis work was to examine 3-dimensional structure as well as the motional properties of two different types of two-domain proteins by solution-state NMR. One of the major tasks of this thesis work was to investigate the domain orientation of these molecules by employing residual dipolar couplings (RDC) which can be used as orientational restraints for increasing the accuracy of protein structures determined by NMR. In the first paper of this thesis two neighboring Epidermal Growth Factor modules (EGF) of Protein S were investigated. Protein S is a down-regulator of blood coagulation and possesses 4 tandem EGF domains. The structure determination of EGF3-4 [Protein Data Bank accession code: 1z65c], employing RDCs, suggests that there is a hinge-like motion in EGF 3, which results in a bending of the structure. This is a unique and so far unprecedented fold of an EGF module. The second paper deals with the structures of human and porcine Beta-Microseminoprotein, MSP, alternatively called Prostatic Secretory Protein of 94 amino acids, PSP94. The structures of the two proteins from these organisms were shown to be very similar [Protein Data Bank accession codes: 2iz3 and 2iz4]. MSP comprises two distinct beta-sheet domains. MSP adopts an extended structure, as determined by employing three different types of RDCs in the structure calculations. This result is in contrast to a recently published structure of porcine MSP (Wang et al. 2005, Journal of Molecular Biology, 346, 1071-1082; Protein Data Bank accession code: 1xhh), where the domains are arranged in a compact conformation. Structure validation showed that the usage of only one type of RDCs cannot solely discriminate between the two different domain orientations. In a subsequent study 15N relaxation data of human and porcine MSP were measured and compared. Model-free analysis of the heteronuclear relaxation data (R1, R2 and steady state NOE of the backbone {1H}-15N) was used to extract information on the dynamics of the proteins. The dynamical information obtained by this method reports on both the overall tumbling and the internal motion of human and porcine MSP. The data show that the human variant of MSP is more flexible than its porcine counterpart. A combination of CPMG and R1,rho experiments was employed to characterize the chemical exchange contribution to R2 for residues of human MSP. It was shown that in human MSP backbone amide nitrogens of at least two residues, Glu31 and Tyr43, experience chemical exchange on a timescale of 0.2 ms. It has recently been shown that human MSP binds to two blood plasma proteins, CRISP-3 ("Cysteine RIch Secretory Protein-3"; Udby et al. 2005, Biochemical and Biophysical Research Communications, 333, 555-561) and PSPBP ("PSP94-Binding Protein"; Reeves et al. 2005, Biochemical Journal, 385, 105-114). Based on the crystal structure of stecrisp, which is a CRISP-3 homologous protein, and NMR data for a 1:1 mixture of human MSP and human CRISP-3, a model for the protein complex is presented in the last paper of this thesis.