Many biological processes rely on specific protein-protein interactions, for example immune responses, cell signaling, transcription, and blood coagulation. Blood coagulation is initiated when a vessel wall is damaged, exposing tissue factor (TF) to the circulating factor VII/factor VIIa (FVII/FVIIa) which results in the formation of the TF:FVIIa complex and thereby the initiation of blood coagulation. One of the substrates for the TF:FVIIa complex is factor X (FX), which is activated to factor Xa (FXa), subsequently leading to a series of reactions resulting in clot formation. Tissue factor pathway inhibitor (TFPI) is the major physiological inhibitor of the sTF:FVIIa complex, involved in regulation of coagulation by forming the TF:FVIIa:FXa:TFPI complex. Occasionally, the blood coagulation mechanism malfunctions, resulting in conditions such as the inability to stop bleeding or thrombosis. The fact that TF is the main initiator of the coagulation makes this an interesting protein to study, in the hunt for means to interfere with players involved in the blood clotting process.

Throughout the studies included in this thesis the site-directed labeling technique is utilized to attach spectroscopic probes to cysteines, introduced at specific positions by mutagenesis, in the protein of interest. These fluorescent or spin-probes are sensitive for changes in their immediate environment and can thus, for example be used to monitor protein-protein complex formation and conformational changes.

No complete structure has been obtained as yet for the large complex involving sTF, FVIIa, FXa, and TFPI. Therefore, we introduced a fluorescent probe at specific positions in soluble tissue factor (sTF) and the changes in fluorescence emission were detected upon sTF:FVIIa:FXa:TFPI complex formation. From these measurements it was concluded that not only parts of the C-terminal domain of sTF (TF2), but also residues in the N-terminal domain (TF1) are involved in binding to FXa in the quaternary complex.

In order to investigate conformational changes occurring in the extended interface between sTF and FVIIa upon binding of different inhibitors spectroscopic probes were introduced in sTF, in the vicinity of the interaction region. From the obtained data it was concluded that the exosite-binding inhibitor E-76 induces equivalent structural changes at the interface of sTF and the protease domain (PD) of FVIIa, as do the active-site inhibitors FFR and TFPI, i.e. makes the region around the active-site more compact. Binding of these inhibitors shows similar effects despite their differences in size, binding site, and inhibitory mechanism.

In addition, the Ca²⁺ dependence of the formation of the sTF:FVIIa complex was studied. Association between sTF and FVIIa during Ca²⁺ titration begins by Ca²⁺ binding to the first EGF-like domain of FVIIa. However, Ca²⁺ saturation of the γ-carboxyglutamic acid-rich (Gla) domain of FVIIa is required for complete sTF:FVIIa complex formation, and we were also able to detect that a Gla domain with vacant Ca²⁺ sites hinders the docking to sTF.

Finally, we investigated the structural changes of free inhibited FVIIa upon sTF and Ca²⁺ binding by FRET and quenching measurements. From this it was concluded that inhibited FVIIa does not seem to undergo large global structural changes upon binding to sTF, when taking the dynamics of free FVIIa into account. However, Ca²⁺ binding induces minor local conformational changes in the active-site region of the PD of inhibited FVIIa and subsequent binding of sTF causesfurther structural rearrangements in this area.

Protein-protein interactions are intrinsic to virtually all cellular processes. However, very little is known about the dynamics of the formation of protein complexes or the order of events that direct the association between two protein molecules (the association pathway), especially when different interacting surfaces are involved. The main objective of the work described in this thesis was to combine a novel probing approach with other well-established techniques togive new insight into structural and dynamic details of a receptor-ligand interaction.

The approach used is based on site-specific labeling, which involves specific introduction of molecular probes that can monitor independent structural and dynamic events both at equilibrium and as a function of time. As a model of protein-protein interactions, we chose the complex formation between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), which initiates the blood coagulation cascade. This is a multi-domain complex that exhibits an extensive binding interface upon formation. Different spectroscopic labels were covalently attached to an engineered cysteine in sTF at positions previously characterized as beeing located in the sTF:FVIIa binding interface. Two spin labels and two fluorescent labels were used, and electron paramagnetic resonance (EPR) and fluorescence emission were monitored to determine the environmental changes sensed by the probe upon formation of the sTF:FVIIa complex. Initially, the properties of the labels and their preferred orientations within the complex were examined and related to the spectral data. This confirmed the tightness of the interaction between FVIIa and sTF, which is comparable to that seen in the interior of globular proteins. The same approach was also used to resolve the contributions of various residues and domains to the global binding energy between sTF and FVIIa. We suggest that the first epidermal growth factor-like (EGF1) domain of FVIIa does not require assistance from the neighboring γ-carboxyglutamicacid (Gla) domain to attain its rigid native interface with sTF. We also monitored conformational changes along the sTF:FVIIa binding interface in the absence and presence of an FVIIa inhibitor. The incorporation of an inhibitor into the active site of the protease domain of FVIIa resulted in tighter binding between sTF and FVIIa only in that particular binding region, leaving the other regions of the binding interface unaffected. Since Ca²⁺ is important for the docking between sTF and FVIIa and most of the Ca²⁺-binding sites are located in the Gla domain, we employed the site-directed labeling approach and subsequent Ca²⁺ titration to specifically monitor the Ca²⁺ -dependent association between sTF and this region of FVIIa. Our approach revealed that occupation of the Ca²⁺ binding site in EGF1 is a prerequisite for forming the sTF:Gla interface. We were also able to resolve two different Ca²⁺ dependent structural rearrangements in the Gla domain essential for the sTF:Gla docking.

Finally, using a combination of stopped-flow fluorescence spectroscopy and surface plasmon resonance measurements, we demonstrated a consecutive binding mechanism for the docking pathway of sTF:FVIIa complex formation. The initial binding of the protease domain of FVIIa to sTF seems to be mediated by an ordered water network and is probably important for rapid formation of the sTF:FVIIa complex.

Proteins are essential participants in virtually all cellular processes. The key to the understanding of the function of a certain protein is a detailed knowledge about its atomic three-dimensional structure. Presently, there is a huge research effort in the search of increased knowledge about the structure and dynamics of protein complexes, as well as in the pursuit of structural information about conformational changes during protein folding and protein aggregation. The main objective of the work described in this thesis was to acquire new structural and dynamic details of relevant proteins in these categories. The methodology used is based on site-directed labeling, which involves specific attachment of molecular probes that are sensitive to their local environment and therefore can be used as reporters of structure and dynamics in proteins. The applicability of the approach was evaluated with reference to already known structural data.

As a model of protein-protein interactions, we have chosen the complex formation between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), which is responsible for the initiation of the blood coagulation cascade. Upon association, an extended, multi-domain binding interface is created between the proteins with a very complex binding pattern. Different spectroscopic labels were covalently attached to an engineered cysteine in sTF at positions previously reported as being located in the sTF:FVIIa binding interface. Two spin labels and two fluorescent labels were used, and their response to the changed local environment upon FVIIa binding was monitored by electron paramagnetic resonance (EPR) and fluorescence spectroscopy, respectively. Initially, the properties of the labels and their preferred orientations within the complex were examined with molecular modeling at a specific site, and subsequently this information was used in the interpretation of the spectral data. The conclusion was a tight interaction between sTF and FVIIa in this region of the complex, in fact comparable to that seen in the interior of globular proteins. In an extended study, we found interactions of similar character at multiple sites not only in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVIIa(sTF:EGF1), but also in the region between sTF and the γ-carboxyglutamic acid (Gla) domainof FVIIa (sTF:Gla). In addition, signs of a tight interaction were found in tlte interface region between sTF and the protease domain (PD) in FVIIa (sTF:PD) in spite of the structural perturbation caused by the attached label. By the same approach we suggest that the EGF1 domain of FVIIa does not require assistance from the neighboring Gla domain to establish a rigid native binding to sTF. Furthermore, the interaction between sTF and EGF1 is largely dictated by Ca²⁺ binding to the site in EGF1. Finally, we monitored conformational changes along the sTF:FVIIa binding interface induced by the incorporation of an inhibitor into the active site of the protease domain of FVIIa. A tighter binding between sTF and FVIIa was detected only in the sTF:PD region, whereas the sTF:EGF1 and sTF:Gla regions were unaffected. The combined use of different spectroscopic techniques and labels (multi-probing) provides valuable complementary information, enabling the comparison of interaction tightness and interaction characteristics, respectively, along the binding interface of a protein complex. The approach also reduces the risk of misinterpretation of data.

The enzyme human carbonic anhydrase II (HCAII) was chosen as a model protein for studies of protein folding and aggregation. HCAII unfolds in a multi-step manner with a molten-globule intermediate state populated between the native and unfolded states. Position 79 in the periphery of the central hydrophobic core of HCAII, was labeled with the same four spectroscopic labels as above. A persistent local cluster associated to the central core was observed in the unfolded state, suggesting an extended residual structure. HCAII is known to form aggregates in its partially unfolded molten-globule intermediate state. We found that the formed aggregates at the site of the labels represent an ensemble of different structures with apolar, compact as well as polar, dynamic regions.

Finally, spin labels can be applied to proteins not only as probes of local structure but also as probes of local polarity. Therefore, a combined theoretical and experimental work was initiated to assess the sensitivity of spin labels such as MTSSL to various solvents and clarify the influence of solvent polarity (dielectric constant, ε) and proticity. We believe that such information can be useful in the interpretation of rigid-limit data from spin-labeled proteins. The g-values g_iso and g_xx as well as the hyperfine coupling constants A_iso and A_zz of the spinlabel were dependent on the solvent properties. At lower polarity (ε<25), the sensitivity of A_iso and A_zz to ε is large, whereas at higher polarity (ε>25), the sensitivity to ε is small, so A_iso and A_zz are instead determined by the proticity of the solvent. From the comparison of experimental and calculated data the propensity of hydrogen bonding of the solvents was estimated. The density functional theory (DFT) method determines the shifts in g_iso and g_xx due to hydrogen bonding more accurately compared to the restricted open-shell Hartree-Fock method.