Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structural characteristics of individual domains in a new context of artificial fusion proteins may help to reveal the rules of internal and external domain communication. Moreover, this could also help explain the mechanism of such communication and address how the mutual allosteric effect plays a role in a such multi-domain protein system. The simple model system of the two-domain fusion protein investigated in this work consisted of a well-folded PDZ3 domain and an artificially designed small protein domain called Tryptophan Cage (TrpCage). Two fusion proteins with swapped domain order were designed to study their structural and functional features as well as their biophysical properties. The proteins composed of PDZ3 and TrpCage, both identical in amino acid sequence but different in composition (PDZ3-TrpCage, TrpCage-PDZ3), were studied using circualr dichroism (CD) spectrometry, analytical ultracentrifugation, and molecular dynamic simulations. The biophysical analysis uncovered different structural and denaturation properties of both studied proteins, revealing their different unfolding pathways and dynamics.

2nd Faculty of Medicine Charles University Prague 5 Czech Republic

CEITEC Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Prague 6 Czech Republic

Department of Biochemistry Faculty of Science Charles University Prague 2 Czech Republic

Department of Structural Biology of Signalling Proteins Division BIOCEV Institute of Physiology Vestec Czech Republic

Faculty of Mathematics and Physics Charles University Prague 2 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague 6 Czech Republic

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