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  • 1.ACDEFGHIKLMNPQRSTVWY Primary structure Secondary structure Tertiary structure Quaternary structure

2. Quaternary structure Refers to the organization of subunits in a protein with multiple subunits Subunits may be identical or different Associate to form dimers (TIM, HIV protease, DNA binding proteins), trimers (MS2 viral capsid protein), tetramers (Haemoglobin, Proteasome, Bacterial photosynthetic reaction center), etc. Subunits have a defined stoichiometry and arrangement Subunits are held together by weak, noncovalent interactions (hydrophobic, electrostatic) Typical Kd for two subunits: 10-8 to 10-16 M (tight association) Entropy loss due to association - unfavorable Entropy gain due to burying of hydrophobic groups - very favourable 3. Why do proteins attain quaternary structures? Morphological function: Many proteins have functions that require creation of large, stable structures. These include long, thin structural elements and large, hollow capsids and rings. Bringing catalytic sites together More complex scaffolds may better support function e.g. by the introduction of a new active site at the interface between subunits. It has been estimated that roughly one sixth of oligomeric enzymes has an active site located at the inter-subunit interface. Stability Larger proteins are more resistant to degradation and denaturation. Indeed, an increase in oligomerization state is one of the protein stabilization strategies observed in thermophilic organisms. Protein stability involves a fine balance between the enthalpic stabilization by many weak nonbonded interactions and the competing effect of various entropic factors of conformational mobility and solvation. 4. Cooperativity (allostery): Allostery and multivalent associations are other functions that create an evolutionary force selecting large proteins with several identical active sites rather than monomeric proteins with a single active site. Reduction of surface area: In general, it is preferable to reduce the protein surface area that is exposed to solvent, by creating a large protein with several identical active sites. Reduction of surface area reduces the amount of solvent needed to hydrate proteins. The reduced surface area provided by an oligomeric protein provides protection from degradation. Reduced surface area also improves the diffusion of substrates to enzyme active sites. 5. Assume that the aqueous cytoplasm is composed entirely of 20 kDa subunits and that all oligomers are spherical in shape. Thus, a monomer would be a sphere of radius 1.8 nm, a dimer would have a radius of 2.2 nm, and so on. Assume that the cytoplasm is 20% protein and that the bound water of hydration is ~1.4 g/g of protein or a hydration shell ~0.6 nm thick. If the aqueous cytoplasm is composed entirely of monomers, the hydrated proteins occupy 47% of the total volume, over twice the 20% volume occupied by the protein alone. Assuming this same 0.6 nm layer of hydration, the volume of the hydrated protein drops to 40% for dimers, 35% for tetramers and 30% for dodecamers. Thus, oligomerization can significantly reduce the amount of water bound to protein surfaces. An analysis of surface area to volume ratio of oligomeric proteins 6. How is large proteins built? The large proteins may be constructed in one of following ways: 1. As long single chains 2. As heterooligomers of several smaller chains 3. As homooligomers of identical chains 7. 1. Error control: By building a large complex from many small subunits, translation errors may be reduced by discarding subunits with defects, providing an extra step for proofreading. 2. Coding efficiency: Homooligomers provide a genetically compact way to encode the information to build a large protein. It reduces the genetic space such as in viruses. 3. Genetic efficiency: Oligomeric proteins may be subjected to amplified evolutionary pressures, as deleterious mutations may be more pronounced and thus removed sooner from the gene pool. Conversely, the advantages of beneficial mutations may also be made evident sooner. 4. Regulation of assembly: Large assemblies built of many identical subunits have attractive regulatory properties, because they are subject to sensitive phase transitions. For instance, actin is involved in many dynamic processes at the cell surface. A collection of actin-binding proteins control the nucleation, growth, termination and disassembly of actin filaments allowing fine spatial and temporal control. What are the advantages of having multimers than large monomers? 8. Protein surface is irregular. What does enable proteins to bind specific molecules? How does oligomerization of proteins occur in the cell? Shape complementarity is necessary for large number of weak interactions and to maximize the strength of interactions ((H-bonds and van der Waals). 9. Finally, oligomerization can arise via fusion of a gene encoding a dimerization or oligomerization domain, such as a coiled-coil domain, onto a previously monomeric protein. 10. Quaternary Structure: Geometry 11. Quaternary Structure: Geometry 12. Protein assemblies built of identical subunits are usually symmetric The homooligomeric proteins found in modern cells are also highly symmetrical with soluble oligomers forming closed complexes related by simple point groups and extended polymers showing helical symmetry. 13. The human growth hormone-receptor complex Asymmetric complexSymmetric complex 14. Why build symmetrical oligomeric proteins? 1. Stability of association: The stability of closed, symmetrical oligomers is a consequence of two factors: (a) the specificity of protein-protein interfaces favors symmetrical complexes, and (b) the maximum numbers of inter subunit interactions are formed in closed complexes. 2. Finite assembly: Proteins must avoid unwanted aggregation. Point group symmetry provides a method to create oligomers of defined copy number. Several disease states seem to be the result of pathological aggregation of mutant proteins such as sickle-cell anemia, Alzheimers disease and prion-related diseases. 3. Folding efficiency: Symmetric protein structures provide fewer kinetic barriers to folding than do asymmetric structures. 15. Where does oligomerization of proteins occur inside the cell? The primary sites for protein synthesis and folding are the cytosol and the endoplasmic reticulum. Cytosolic proteins are synthesized, folded and oligomerized in the cytosol. Membrane and secretory proteins are synthesized in the endoplasmic reticulum (ER) and oligomerization typically occurs within the ER, although, in some cases, oligomerization takes place in the intermediate compartment and Golgi apparatus. 16. What are the factors which can affect the oligomer formation? 1. Ligand binding: Many receptors undergo dimerization upon ligand binding. 2. Polymerization: Proteins such as actin can polymerize. Other proteins can polymerize after undergoing a conformational change giving rise to amyloid fibrils. 3. Concentration: The oligomerization state of a protein depends on the concentration of protein. At nM concentration the tendency to be in the monomeric state will be much higher than in the M or mM range. 4. Environmental condition: Weak associations may happen due to conditions such as concentration, temperature, pH, solvent conditions (the ionic strength, metal cofactors and effectors concentrations) and have higher Kd values in the M or mM range. 5. Domain linker: it is known that variation of inter-domain linker lengths can result in variations in oligomeric state. Some examples are the legume lectins, which can dimerize by various modes as well as tetramerize, the cystine-knot growth factors and lumazine synthase. 17. How to determine the oligomeric states of proteins experimentally? In general, the following in vitro biophysical techniques can be used: 1. Size exclusion chromatography 2. Cross-linking 3. Analytical ultracentrifugation 4. Isothermal titration calorimetry 5. Mass spectrometry 6. Frster resonance energy transfer (FRET) 7. Scattering techniques 8. Yeast two hybrid assays 9. Fluorescence anisotropy 10. NMR spectroscopy 18. Characteristics of oligomeric interfaces Interfacial residues tend to protrude from the surface of the protein and the interaction surface tends to be circular in shape. Proteinprotein interaction interfaces are relatively planar as are many hetero-oligomer interfaces. The buried surface area in obligate homodimeric proteins is usually greater than 1400 2. In nonobligate complexes, the interface buried surface area is usually less than 2500 2, whereas for weak and transient associations the buried surface area of the interface is less than 1000 2 . It has been found that certain conserved residues or hot spots generally at the center of an interface are responsible for most of the binding energy of an oligomeric interaction. 19. Inter-subunit interfaces are less non-polar, and have a greater proportion of hydrophilic and polar residues, than a typical protein hydrophobic core. Approximately one-fifth of the residues at oligomeric interfaces are polar, a greater proportion than is found in buried hydrophobic cores. Hydrogen bonds and salt bridges are important for the stabilization of oligomeric interfaces, as suggested by the prevalence of polar hot spot residues. Early studies suggested that there is about one hydrogen bond per 200 2 of subunit interface. Oligomeric interfaces often have significant electrostatic and geometrical shape complementarity that gives rise to the specificity of the interaction. 20. It has been calculated that the average oligomeric state of cellular proteins is tetrameric and a survey suggests that 35% or more of the proteins in a cell are oligomeric. Most oligomeric proteins are homo-oligomers. Higher-order oligomers are less prevalent and a relatively small fraction of oligomeric structures have odd numbered stoichiometries. M