BS2520 - Protein Structure and Function Lecture 6 Protein Engineering Why would we want to engineer proteins? To study protein functions - to change/modify structure of native proteins to study their function To improve protein stability, solubility and overcome expression limitations To improve protein functions - Enzymes and enzyme inhibitors - better enzymes, e.g. more stable/efficient DNA polymerases (thermostable/PCR) - more efficient enzymes (or inhibitors), better catalytic function (biotechnology applications) - new enzymes, e.g. with higher processivity, new metabolic pathways - new enzyme drugs for therapeutic applications To create new functional proteins - Therapeutic proteins and therapeutic antibodies - unconjugated antibodies (Fc attracts macrophages) - radioactively tagged antibodies (chemical modification) - toxin conjugated (recombinant) - enzyme conjugated antibody (recombinant) - Regulatory proteins (e.g. transcription factors) - to be able to regulate expression of genes (up/down)-> cure disease (zinc-finger proteins, alternative to small drugs) - New biosensors and diagnostics (devise proteins to fit existing technologies) - Many more example exist (search Web of Science using the phrase "Protein Engineering") Engineering Protein Stability Increase the stability of proteins by optimising structure and folding. This is rational engineering and design of protein structure and function. A successful attempt was to introduce disulfide bridges to improve structure - the engineered disulfide bridge was between residues 21 and 142 of a phage T4 lysozyme - this substantially enhanced the stability of the protein without making the folded structure more rigid - the disulfide increased the melting temperature of the protein by 11 degrees C - Matsumura et al. Alpha helix: - Stabilising dipoles fidue to orientation of hydrogen bonds) of alpha-helices fiby putting opposite charges on either end of the dipole) increases stability - if we add a basic AA to the N-terminus and an acidic AA to the C-terminus, these charges will compensate the dipole and therefore, stabilise the helix. - Coiled-coils = alpha helices bound together through the interaction between hydrophobic side chains which occur every 7 residues - heptad repeats. - We can increase the stability of the folding between two alpha helices by making sure that non-polar AA fill these positions - we can easily engineer this structure. - If we introduce hydrophobic side chains at every 7th position, these helices will form a coiled-coil.
Beta sheets: - If we know that side chains come together, and if not involved in any other structural/functional interaction, then they might pair basic with acidic side chains - this will improve the stability of the strand. - Very often, beta strands occur in beta sandwiches - in this case, there are 2 beta sheets placed against each other and so some of the side chains will be facing between the 2 sheets, and some will be facing outside. - This structure relies on hydrophobic, non-polar interactions between the side chains between the 2 sheets. - If there were polar side chains here, we can replace them with non-polar ones to increase stability. - On the other hand, increasing the number of polar side chains