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.

• Keywords
• chimeras, fusion protein, protein domains, protein dynamic studies,
• MeSH
• PDZ Domains * genetics   physiology   MeSH
• Recombinant Fusion Proteins * chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Molecular Dynamics Simulation MeSH
• Tryptophan * chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Recombinant Fusion Proteins * MeSH
• Tryptophan * MeSH

Many carbohydrate-binding proteins contain aromatic amino acid residues in their binding sites. These residues interact with carbohydrates in a stacking geometry via CH/π interactions. These interactions can be found in carbohydrate-binding proteins, including lectins, enzymes and carbohydrate transporters. Besides this, many non-protein aromatic molecules (natural as well as artificial) can bind saccharides using these interactions. Recent computational and experimental studies have shown that carbohydrate-aromatic CH/π interactions are dispersion interactions, tuned by electrostatics and partially stabilized by a hydrophobic effect in solvated systems.

• Keywords
• CH/π interactions, carbohydrate-protein interactions, interaction energy, lectins, non-canonical hydrogen bond,
• MeSH
• Lectins chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Carbohydrates chemistry   MeSH
• Protein Binding MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Lectins MeSH
• Carbohydrates MeSH

Combining computational and experimental tools, we present a new strategy for designing high affinity variants of a binding protein. The affinity is increased by mutating residues not at the interface, but at positions lining internal cavities of one of the interacting molecules. Filling the cavities lowers flexibility of the binding protein, possibly reducing entropic penalty of binding. The approach was tested using the interferon-γ receptor 1 (IFNγR1) complex with IFNγ as a model. Mutations were selected from 52 amino acid positions lining the IFNγR1 internal cavities by using a protocol based on FoldX prediction of free energy changes. The final four mutations filling the IFNγR1 cavities and potentially improving the affinity to IFNγ were expressed, purified, and refolded, and their affinity towards IFNγ was measured by SPR. While individual cavity mutations yielded receptor constructs exhibiting only slight increase of affinity compared to WT, combinations of these mutations with previously characterized variant N96W led to a significant sevenfold increase. The affinity increase in the high affinity receptor variant N96W+V35L is linked to the restriction of its molecular fluctuations in the unbound state. The results demonstrate that mutating cavity residues is a viable strategy for designing protein variants with increased affinity.

• MeSH
• Interferon-gamma chemistry   metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Models, Molecular * MeSH
• Interferon gamma Receptor MeSH
• Receptors, Interferon chemistry   genetics   metabolism   MeSH
• Protein Folding * MeSH
• Amino Acid Substitution * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• IFNG protein, human MeSH Browser
• Interferon-gamma MeSH
• Receptors, Interferon MeSH