lec06.docx

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Department
Biological Sciences
Course
BIOC12H3
Professor
Shelley A.Brunt
Semester
Fall

Description
lec06 lecture outline 1. protein folding 2. protein complexes 3. chaperones quaternary structure 1. individual subunits in tertiary structure are not nearly as stable as they are when in quaternary structure a. multimeric (oligomeric) proteins have two or more polypeptide chains which provide stability b. identical to protomers i. what are protomers? 1. structural unit of a multiunit (oligomeric) protein 2. multimeric nature is critical to how proteins function a. critical to mechanisms for regulating their function 3. different subunits given greek letter representations 4. multimeric proteins do not have to contain different polypeptides 5. structure held together by weak non-covalent bonds a. primarily hydrophobic b. tight association also allows separation of subunits when needed i. quaternary structure is more stable than subunits ii. increases half life c. contact regions resemble interior of a single subunit 6. stability becomes critical, but folding has to be built into structure 7. when we want protein to change structure slightly, it needs to be able to come apart in some ways a. this is where non-covalent bonds come in b. the way in which subunits come together i. the folding of the subunit itself (the center of the polypeptide is hydrophobic) 1. same thing occurs in the quaternary structure a. the center of protein is generally hydrophobic 8. substrate binding site is built when subunits come together a. binding to a cofactor changes structure of protein i. different proteins can share same subunits 1. subunits for ligand binding a. what is a ligand? i. molecule that forms a complex with a biological molecule for a biological purpose b. active sites of some oligomeric enzymes are formed from residues from different polypeptide chains i. the 3D structure changes with ligand binding chicken triose phosphate isomerase 1. two identical subunits with alpha / beta barrel folds a. example of an oligomeric / multimeric protein that has two identical subunits tubulin 2. example of multimeric protein structure a. alpha beta subunits come together to form tubulin hetero dimers i. tubulin added to + end and lost at - end 1. this complex (multimeric) structure consists of only two subunits a2b2 tetramer of human haemoglobin 1. consists of alpha and beta protomers a. tetramer subunits come together to form subunit (heme) binding sites b. substrate is oxygen schematic diagram of an immunoglobulin (IgG) 2. IgG is one class of immunoglobin a. 4 heavy chains / 2 light chains form a "Y" shape b. FC region will interact with FC receptor i. what is FC? 1. fragment crystallization region a. tail region of antibody that interacts with cell surface receptors (FC receptors) b. allows antibodies to activate immune system c. composed of two identical protein fragments 2. function a. binds to receptors b. mediates different physiological effects of antibodies i. immune cell activity c. ensures antibody generates an appropriate immune response for a given antigen ii. notice the disulfide bonds in yellow 1. between heavy chains 2. between heavy chain with light chains 3. where do we find immunoglobulin? a. circulating outside the cell i. extracellular b. not a good environment for proteins i. disulfide bonds are therefore important to stabilize this protein 1. formation of disulfide bonds is oxidation reduction a. cytosol has highly reduced environment so it is hard for disulfide bonds to form inside the cell 4. structure of antigen binding site is predominantly beta sheets which stabilizes the structure a. why does beta sheets stabilize structure better than alpha helices? i. interchain h bonds between polypeptide chains natural occurrence of oligomeric proteins in e. coli 1. 1/5 of proteins made in e. coli form tetramers 2. as oligomeric state increases, percent of that particular state of protein decreases a. however, higher oligomers are imporant, and do not follow this pattern i. functional advantages to oligomerization 1. more complex scaffolds to better support function 2. allosteric regulation  additional level of control 3. greater likelihood of error free transcription in small protein sequence subunits that come together to form a large protein 4. larger proteins more resistant to degradation and denaturation macromolecular assemblies 1. highest level of protein structure a. e.g. i. ribosomes ii. spliceosomes iii. nuclear pore complex 1. composed of multiple proteins to form a cage for molecules to move in and out as long as they have an appropriate signal to the pores iv. transcription initiation complex v. chaperonins 1. very unique structures that resides within cytosol and mitochondria to help the folding of proteins vi. proteosomes 1. similar structure to chaperonin but instead decreases proteins 2. macromolecule assemblies are important large protein complexes in mycoplasma pneumonia 1. polypeptides come together to make functioning protein unit a. how proteins function in cell i. most transcription factors require dimers 1. i.e. not single proteins ii. most proteins are not working by themselves 1. even when they are working by themselves, they require other proteins to fold them and get them where they are, location wise a. chaperonins help with folding and transport bacterial flagellum 1. 21 core subunits in all species go into creating the structure of a bacterial flagellum e.coli interactome 2. interactomes a. knowledge we know of protein sequences b. a map of which proteins are acting with which proteins 3. main point a. individual proteins interact with many other proteins i. not interacting ALL the time, but still interacting with many different proteins b. these maps helps us understand which proteins 'play' with which other proteins in the cell protein stability and folding 1. protein has to be folded and stable a. how do we form an alpha helix organized in tertiary structure i. don't know answer, but know models to suggest a method 1. electrostatic interactions have some role a. but if protein exposed to water, this role is greatly decreased because water dissolves the component ionic interactions b. many proteins interact with different metals to stabilize structure i. introduction of co-factors 2. hydrophobic interactions has the greatest effect a. hydrogen bonds critical in structure contribute less to stability than the hydrophobic effect b. electrostatic interactions contribute to some degree c. disulfide bonds i. cross link extracellular proteins 1. what does this mean? a. A cross link is a bond that links one polymer chain to another b. They can be covalent bonds or ionic bonds c. Proteins naturally present in the body can contain crosslinks generated by enzyme catalyzed or spontaneous reactions d. Cross links are important in generating mechanically stable structures such as i. Hair ii. Skin iii. Cartilage e. Disulfide bond formation is one of the most common cross links d. metal ions can stabilize structure hydropathy plot of bovine chymotrypsinogen 1. hydrophobic  interior of protein 2. hydrophilic  exterior of protein 3. classic role of zinc in the structure of the 'zinc finger' structure which is critical in protein-protein interactions, a class of transcription factors 3D structure 1. the amino acid sequence of a protein determines its 3D structure a. based on work on ribonuclease A (RNase A) i. key in understanding denaturation / renaturation b. don't know how sequence determines 3D structure, but it does conversion of native conformation with loss of biological activity is denaturation 2. thermal denaturation curve of RNase A a. chemical or environmental changes b. amount of energy needed is low i. equivalent of breaking 3-4 h bonds ii. destabilization of just a few weak interactions leads to complete loss of native conformation 1. very few bonds need to be broken before protein starts to unfold and lose its activity c. denaturation curve monitored by i. UV absorption ii. viscosity iii. optical density 3. RNase A a. very stable structure b. 50% unfolded range denatures around 30-32 degree range i. Why is 50% unfolded at this temperature? c. denaturation is not the same as degradation i. denaturation vs degradation 1. denaturation a. unfold proteins 2. degradation a. decompose protein into individual amino acids ii. important distinction 4. some proteins may completely unfold following denaturation a. forming random coil i. completely disordered b. others may retain some internal structure i. but function will still be lost chaotropic agents denature proteins into random-coils 1. denature proteins into random coils by allowing water molecules to solvate nonpolar groups in interior of proteins a. normally these nonpolar interactions stabilize proteins i. e.g. urea and guanidinium chloride 1. will denature proteins into random coils 2. significant enough to make it lose its function 2. renaturation / denaturation studies with SDS not efficient because SDS is hard to remove afterwards a. instead use chaotropic agents such as urea / guanidium chloride i. what are chaotropic agents? 1. Chaotropic agents are substances which disrupts the structure of and denatures macromolecules such as proteins and nucleic acids hydrophobic tails of detergents also denature proteins by disrupting hydrophobic interactions 1. How do the hydrophobic tails disrupt hydrophobic interactions? native conformation is sometimes stabilized by disulfide bonds 1. disulfide bonds often found in secreted proteins (extracellular) a. e.g. RNase A b. disulfide bonds allow the protein to resist unfolding / degradation i. important in proteins in the extracellular environment 2. incorrect disulfide bonds forming can be prevented a. how? 3. important for midterm a. important to remember disulfide bonds form AFTER folding i. i.e. once the cysteine residues have been brought into close proximity b. requires oxidation of the thiol groups on the cysteines i. forming cystine 4. moment you go into extracellular region of the cell, the cell loses control of protein a. disulfide bonds help to stabilize proteins outside the cell 5. breaking disulfide bonds requires a. reduction of the thiol groups on the cysteines in addition to the disruption of hydrophobic interactions and h bonds 6. cytoplasm is a reducing environment a. extracellular is a oxidizing environment with less control of temperature and pH role of beta-mercaptoethanol in reducing disulfide bonds 1. beta-mercaptoethanol helps reduce disulfide bonds which result in denaturing of protein a. urea breaks h bonds and hydrophobic bonds b. mercaptoethanol breaks disulfide bonds 2. treatment with an 8M urea solution containing beta mercaptoethanol completely denatures most proteins a. the urea breaks intramolecular hydrogen and hydrophobic bonds b. mercaptoethanol reduces each disulfide bridge (-S-S-)into two sulfhydryl (-SH) groups c. when these chemicals are removed by dialysis i. the -SH groups on the unfolded chain oxidize spontaneously to reform disulfide bridges ii. the polypeptide chain simultaneously refolds into its native conformation protein denaturation and refolding of RNase A 1. the protein is normally folded into its native conformation a. which contains four disulfide bonds 2. if incorrect disulfide bonds form a. can denature it i. which will result in spontaneous reformation of the correct conformation 1. usually 3. main point of slide 24? denaturation / renaturation cycle of RNase A 1. proteins not properly folded a. results in incorrect disulfide bonds forming within the cell b. scenario where incorrect disulfide bonds form because of incorrectly folded proteins c. proteins sometimes incorrectly fold and form inappropraite disulfide bridges inside the cell (in vivo) i. enzyme called protein disulfide isomerase (PDI) catalyzes the reduction of incorrect disulfide bonds to allow correct folding 1. rearranges non-native disulfide bonds by binding to inappropriate ones 2. catalyzes the correct formation leading up to native conformation 2. chaperonins helps fold proteins 3. proteosomes help degrade proteins that cannot be fixed by chaperonins a. aggregates of misfolded proteins lead to neurological diseases such as parkinsons and alzheimers how do proteins get folded? 1. cooperative process a. gene expression (DNA  RNA  protein) and protein folding i. transcription  translation ii. DNA  amino acids  unfolded state  folding intermediate  native state  protein structure and function b. protein complexes critical to function i. RNA polymerase ii. ribosomes c. as proteins being made, it is immediately protected by chaperonins i. chaperonins fold it and move it goal: protein folds into a compact conformation 2. the polar amino acid side chains tend to gather on the outside of the protein a. where they interact with water via h bonds 3. the nonpolar amino acid side chains are buried on the interior to form a tightly packed hydrophobic core of atoms that are hidden from water a. hydrophobic core region contains nonpolar side chains 4. this model is done assuming no assistance from chaperonins a. proteins fold with hydrophobic elements toward the core, protected from water 5. protein folding depends on cooperativity of folding a. i.e. formation of one part of the structure leads to correct folding of other parts of the structure b. as it folds, it adopts lower and lower energy forms i. falling into an energy well 1. minimum energy is at the bottom of the well 2. once folded, it is much less susceptible to degradation than when in an extended form 6. what is an energy well? a. energy is needed at beginning of folding event i. it is much greater at the beginning than the subsequent folding events b. concept of energy well i. maximum energy used at the top ii. minimum energy used at the bottom c. example of energy well for protein folding i. funnel represent free energy potential of folding protein 1. not very realistic because it doesnt take into account the folding and unfolding attempts at native conformation ii. protein folds and unfolds and folds again until you reach native form of protein 1. as suggested by energy well diagram on the right with peaks and dips before hitting the bottom of the energy well 7. proteins need to be folded really quickly current view of protein folding 1. highly cooperative process 2. each domain of a newly synthesized protein rapidly attains a 'molten globule' state a. original folding in on its core into molten globule is the first step in folding process b. what is a molten globule? i. Read on 3. subsequent folding occurs more slowly and by multiple pathways a. often involving the help of a molecular chaperone i. some molecules may still fail to fold correctly 1. these are recognized and degraded by specific proteases 4. three stages in unassisted protein folding a. i.e. without chaperonin i. small proteins can fold on its own 1. but most proteins in cells are relatively large ii. unassisted proteins 1. molecules condenses along hydrophobic core into a molten globule 2. much of the secondary structure is formed and present at this point 3. long range interactions then form the tertiary structure that leads to the native 3D conformation b. in its denatured state, the entire polypeptide chain assumes a random conformation i. under appropriate refolding conditions, the molecule condenses around a hydrophobic core into a 'molten globule' 1. molten globule is a a. compact b. but non-native c. intermediate 2. in this folding intermediate, much of the secondary structure is present 3. long range interactions then form the tertiary structure a. folding the molecule into its native 3D conformation generalized view of protein folding 1. sequence collapses on itself with hydrophobic regions internalized a. unfolded  molten globule  native conformation generalized mechanism of folding 2. folding is very rapid a. less than a second 3. in its unfolded state, there are more than 10 100possible conformations a. final folded state depends on noncovalent forces such as i. hydrophobic effect ii. h bonding iii. van der waals interactions iv. charge charge interactions b. together they provide stability to the native conformation c. remember the weakness of the individual interactions allow small conformational changes 4. reality is that protein in unfolded states has many possible conformations a. but all conformations meet at an intermediate called the molten globule i. the result is a folded state in which noncovalent forces provide stability to native formation as an aggregate 1. but the individual bonds are still weak 5. however, no actual protein folding pathway is fully described yet a. key point is the intermediates in protein folding pathways b. prediction of 3D structure from sequences remain difficult models for protein folding 1. three main models a. framework model i. secondary structure forms ii. assembly of secondary structure into folded conformation iii. focus on secondary structure b. hydrophobic collapse model i. formation of molten globule ii. growth of secondary structure iii. focus on tertiary structure c. nucleation-condensation mechanism model i. secondary and tertiary structures of the protein are made at the same time ii. hierarchical assembly hypothetical protein-folding pathways 2. molten globule concept is important a. folding inward due to hydrophobic effect i. AKA hydrophobic collapse b. must be as a result of primary sequence i. therefore primary sequence should predict native conformation 1. but we dont know how c. h bonds and hydrophobic interactions are important i. hydrophobic effect 1. collapsing onto itself 2. internalizing the hydrophobic region 3. most polar regions remain in contact with water at the surface 4. some polar side groups forced into the interior by countering the polarity with the formation of h bonds a. the start of secondary structure such as amphipathic strucutures d. primary sequences of proteins i. extended protei
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