The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxy-glutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.

The specific complex between the extracellular part of tissue factor (sTF) and factor Vlla (FVlla) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVlla interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma -carboxyglutamic acid (Gla) domain of FVlla, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVlla. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVlla binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVlla, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2.2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)ethyl)amino)naphtalene-1 -sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation, HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 Angstrom from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 Angstrom from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At similar to1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 Angstrom in diameter depending on the experimental conditions and spectroscopic technique used.

The isotropic and anisotropic hyperfine coupling constants and g-values of the nitroxide spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (MTSSL) were determined from 9-GHz and 95-GHz electron paramagnetic resonance (EPR) measurements in various solvents with a large distribution in polarity and proticity. The parameters Aiso, giso, Azz, and gxx of MTSSL were found to be sensitive to changes in solvent properties, where A-values increased and g-values decreased due to increased solvent polarity or proticity. A linear correlation was found for the isotropic (giso, Aiso) and anisotropic (gxx, Azz) parameters, respectively. Furthermore, density functional theory (DFT) calculations of the same parameters were performed for a model spin label with the possibility to vary the dielectric constant (e) of the medium and the number of hydrogen bonds formed with the nitroxide oxygen. From a qualitative analysis of experimental and calculated results, it was possible to specify the causes of the parameter shifts in more detail. In the "apolar region" (e < 25), the sensitivity of Aiso and Azz to e is large. However, in the "polar region" (e > 25), the sensitivity to epsi, is small, and the shifts in Aiso and Azz are mainly determined by the proticity of the solvent. Methanol was found to form ~1 and water ~2 hydrogen bonds to the nitroxide on average. The DFT method determined the shifts in giso and gxx due to hydrogen bonding more accurately compared with the restricted open-shell Hartree-Fock method. The anisotropic spin label-solvent data can be used in the interpretation of rigid-limit data from spin-labeled proteins to gain further insight in local environmental properties.