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DBT2117: Biochemistry (I) Lecture 12 Protein folding Quaternary structure 1 Disruption of Protein structures – denaturation • Under harsh conditions, a protein loses its functional 3‐D structure. • This process is called denaturation. • Denaturing conditions include: • Increased temperature • pH becomes extremely acidic or alkaline • Organic solvents or urea (chaotropic agents: disrupting H‐bonding between water, reducing hydrophobic effect) *note urea can solubilize denatured proteins 國立交通大學生物科技學系 蘭宜錚老師 1 Protein folding How do proteins know what secondary/tertiary structures to form? • Most of the information for determining the 3‐D structure of a protein is carried in the amino acid sequence of that protein • Chris Anginsen RNase A refolding experiment showed that denaturation and refolding processes can be reversible. (Nobel prize in 1972) Incorrectly folded protein due to random disulfide bonds – only 1% of these molecules are active (because 8 Cys can have 105 possible different combinations) Here urea is used to denature and solubilize the protein Denaturation of proteins can be monitored Thermal denaturation of RNase A: • Differences between the native and denatured conformations can be detected by several spectroscopic methods. • This graph shows the fraction of protein that is denatured as measured by: • increase in solution viscosity • change in optical rotation at 365 nm • change in UV absorbance at 287 nm • All 3 techniques indicate the same fraction unfolding as a function of increasing temperature. 國立交通大學生物科技學系 蘭宜錚老師 2 Folding of globular protein is thermodynamically favorable • • • The folding of a globular protein is clearly a thermodynamically favorable process under physiological conditions. The overall free energy change for folding must be negative. This negative free energy change is achieved by a balance of several thermodynamic factors: • Conformational Entropy – entropy decreases when proteins fold random coil (higher entropy) folded protein (lower entropy) • Charge–Charge Interactions – stabilizing when opposite charges are near when pH is too high or too low, charges and attractions are lost – denature. • Internal Hydrogen Bonds – stabilizing H‐bonds form between polar R‐groups • van der Waals Interactions – stabilizing attraction as globular proteins are well packed. • Hydrophobic effect Overall balance in thermodynamics for protein folding The stability of the folded structure of a globular protein depends on the interplay of three factors: • The unfavorable conformational entropy change, which favors the unfolded state. • The favorable enthalpy contribution arising from intramolecular interactions. • The favorable entropy change arising from the burying of hydrophobic groups within the molecule. 國立交通大學生物科技學系 蘭宜錚老師 3 Disulfide bond Disulfide bonds are not essential for refolding (as demonstrated in the RNAse experiment), however they provide stability to the structure once folded. • Some folded proteins are stabilized by internal disulfide bonds, in addition to noncovalent forces. • Disulfide bonds bonds are relatively rare and are found primarily in proteins that are exported from cells, such as ribonuclease, BPTI, and insulin. • One explanation is that the environment inside most cells is reducing and tends to keep sulfhydryl groups in the reduced state. • External environments, for the most part, are oxidizing and stabilize bridges. Thermal denaturation of BPTI: • The percent denaturation as a function of temperature at pH 2.1 is indicated for the native protein and for the protein in which the Cys 14–Cys 38 disulfide bond has been reduced and carboxymethylated. Kinetics of protein folding • The folding of globular proteins from their denatured conformations is a remarkably rapid process, often complete in less than a second. (example: E. coli can make 100 amino acid residue polypeptide in about 5 seconds at 37 C) • Protein folding is not a completely random search through a vast conformational space. • Rapid kinetic studies, using a variety of physical techniques to monitor different aspects of protein structure, show that folding takes place through a series of intermediate states. A simplified representation of the folding pathway for a protein: •“U” is the unfolded or denatured state. •“F” is the folded or native state. •“I” “on‐pathway” are intermediate states. •Off‐pathway states include aggregates and other non‐native states that may be kinetic or thermodynamic “dead‐ends” •In fact, not all pathways leading to such states are irreversible. 國立交通大學生物科技學系 蘭宜錚老師 4 Off pathway states • Folding can be delayed by trapping of molecules in “off‐pathway” states. • One of the most common of folding errors results from the incorrect cis–trans isomerization of the amide bond adjacent to a proline residue: • Trans conformation is more stable than cis (4:1) • Conversion of cis to trans by an enzyme call prolyl isomerase speeds up folding in vivo Folding funnel model As folding protein follows a “downhill” trajectory on the energy landscape, the funnel becomes narrower, restricting the number of conformations accessible to the protein. Molten globule states Partially folded states that have some native‐ like seoncdary structures, but lacking defined tertiary 國立交通大學生物科技學系 蘭宜錚老師 “multistate” because of metastable intermediates (local minima) 5 Protein topology and folding rates • Different primary sequence can have similar topologies. • The two SH3 domains fold with similar rates, as do ADAh2 and acyl phosphatase, suggesting that protein topology is an important factor in determining the rate of protein folding. • Examples of low and high contact order (sequence separation between residues that physically contact in the native state). • Proteins with higher contact orders tend to fold with slower rates. Molecular chaperones • While many proteins can fold themselves, some proteins require the action of specialized proteins called molecular chaperones to achieve proper folding. • Molecular chaperones functions to keep the newly formed protein out of trouble. • improper folding • aggregation • Improper folding results from intermolecular contacts (such as exposed hydrophobic groups aggregating with other polypeptide strands) 國立交通大學生物科技學系 蘭宜錚老師 6 Molecular chaperone GroES and GroEL The cavity of GroEL‐GroES complex (chaperonins) provides a favorable environment in which non‐native proteins can fold properly Many chaperone proteins are also heat shock proteins Which help refold denatured enzyme upon increased temperature 1. Unfolded protein line 2. ATP and GroES binds up with Hydrophobic and changes GroEL residues conformation 3. Conformation change lead to hydrophilic/ negative surfaces 4. Folded protein leaves GroEL‐ES Protein misfolding Protein misfolding is the basis for several diseases, including Alzheimer’s disease and Parkinson’s disease. In human, most of these diseases are associated with the formation of highly ordered protein aggregates called amyloid fibrils or plaques 國立交通大學生物科技學系 蘭宜錚老師 7 Amyloid fibrils Prion diseases Prions – Proteinaceous infectious particles (proteins) Prions cause neurodegenerative diseases. (transmitted independent of DNA or RNA) • Prion related protein (PrPC) is a normal protein found in cells. • PrPC has chances to spontaneously form PrPSC, the infectious prion. • PrPSC interacts with normal PrPC and turn them into PrPSC. • PrPSC is super stable – can even tolerate very high temperatures PrPC PrPSC • Prions cause mad cow disease, scrapie in sheep, kuru in humans, etc 國立交通大學生物科技學系 蘭宜錚老師 8 Structure prediction • Protein secondary structure can now be predicted with good accuracy. Tertiary structure prediction is not as accurate, but can also be done with good approximation. • The more known structures we have, the more accurate we can design our software and models to predict protein structures. • Many computational algorithms have been developed for protein prediction. • One simple approach is to look at the frequency of amino acid residues that favor particle secondary structures. Structure prediction – tertiary structures In prediction of tertiary structures, algorithm seeks the global energy minimum • this calculation is based on quantum mechanics and assumptions about solvent interactions • as the program looks for global energy minimum. Its search method must be smart. Otherwise, it will waste a lot of time looking at the vast possible conformation that a protein can adopt Rosetta – an algorithm designed to predict protein structure. • First, fragments of the protein sequence are organized into local short structure motifs. This is done by comparing sequences to a structural library. • Then, these short fragments are packed together in ways that give low‐energy conformations, ignoring side chain details (only look at charge and polarity). • Lastly, the details of the side chains are added back and calculation optimizes energy minimum 國立交通大學生物科技學系 蘭宜錚老師 9 Online prediction tools http://robetta.bakerlab.org/ Quaternary structures The four levels of protein structure: Homotetramer Homotypic – association between identical polypeptide chains Heterotypic – association between subunits of very different protein structures. Multiple subunits join together: Dimer, Trimer, Tetramer, Pentamer, Hexamer, Heptamer, Octamer…. So on 國立交通大學生物科技學系 蘭宜錚老師 10 Quaternary structures • The interactions between the folded polypeptide chains in multisubunit proteins are of the same kinds that stabilize tertiary structure • • • • • salt bridges hydrogen bonding van der Waals forces the hydrophobic effect disulfide bonding • These interactions provide the energy to stabilize the multisubunit structure. • Association of polypeptide chains to form specific multisubunit structures is the quaternary level of protein organization. Quaternary structures ‐ interactions Heterologous interaction – the interacting groups lie in different regions of the subunit Isologous interaction – symmetrical and identical interactions between the subunits 國立交通大學生物科技學系 蘭宜錚老師 11 Quaternary structures ‐ symmetry • Although composed of asymmetric polypeptides, proteins adopt many symmetrical patterns in forming quaternary structures. • Most proteins assemble into point group symmetry (shown on the right). • n‐fold axis of symmetry corresponds to a rotation of 360/n to its neighbor • Some proteins assemble into helical structures • Additional isologous interactions can give rise to more levels of symmetry (such as the Dn symmetry, cubic, icosahedral) Quaternary structures – asymmetrical Heterotypic Protein–Protein Interactions: •A dimer without symmetry. o Two subunits can associate by heterologous interactions and still not yield an indefinite chain. o The interaction sites (A, B, C, D) are blocked from reaction with more monomers by the close fit of the surfaces. Interaction of BPTI with trypsin: • The BPTI molecule fits snugly onto the surface of the trypsin molecule (blue), blocking the active site of trypsin. 國立交通大學生物科技學系 蘭宜錚老師 12 Quaternary structures Beside each electron micrograph is a diagrammatic representation of the helical aggregate structure. (a)Actin (b)Tobacco mosaic virus. • In the virus, protein subunits form a helical array about a helically coiled RNA. Viral capsid proteins are often icosahedral As it maximize internal space. Protein folding https://www.youtube.com/v/q7sloonEE5k 國立交通大學生物科技學系 蘭宜錚老師 13