Studies of Local Interactions between and within Proteins using Site-Directed Labeling Techniques

Abstract: 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 giso and gxx as well as the hyperfine coupling constants Aiso and Azz of the spinlabel were dependent on the solvent properties. At lower polarity (ε<25), the sensitivity of Aiso and Azz to ε is large, whereas at higher polarity (ε>25), the sensitivity to ε is small, so Aiso and Azz 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 giso and gxx due to hydrogen bonding more accurately compared to the restricted open-shell Hartree-Fock method.

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