chapter 3- november 1 proteins highlights structure and chemistry of amino acids linkage to form a...
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CHAPTER 3- November 1Proteins
HIGHLIGHTS
Structure and Chemistry of amino acidsLinkage to form a polypeptide monomer to polymerForces that guide foldingModifications and degradationFunctional designCommon techniques
Function depends on the structure
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FUNCTIONALLY VERY DIVERSE:
Bind ions, nuc acids, other proteins, CHO
Catalyze numerous reactions
Provide structural rigidity
Control flow and conc across plasma membrane
Sensors / switches / gene expression
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COOH Amino acids (monomeric subunits) H - C - NH2 R, n=20
– determine its properties
R - Diversity peptide of 4aa has 204 possible or 160,000 sequences
Amino acids are the building blocks of Proteins
• 20 Different amino acids (a.a.) - Alphabet
• unbranched, linear chains of a.a.
• correct 3-D structure is essential for function
•Monomer= amino acid; polymer=peptide or polypeptide
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R Chains (Special Properties)
• Hydrophilic (surface) - Basic +ve lys(K), arg(R), (His) - Acidic -ve glu(E), asp(D) - polar Ser, Thr, asn(N), gln(Q)
• Hydrophobic (core) Ala, Val, Ile, Leu, Met
phe(F), tyr(Y), trp(W)
• Special Cys, Gly, Pro
Polarity is a critical feature for shaping 3D structure
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Special:
Alanine ala A CH3-CH(NH2)-COOH
Glycine gly G NH2-CH2-COOH too flexible, fit tight spaces
Cysteine** cys C HS-CH2-CH(NH2)-COOH Sulfhyrdal group (disulfide bond or bridge)
Proline pro P NH-(CH2)3-CH-COOH kink, cyclic ring, rigid
Isoleucine ile I CH3-CH2-CH(CH3)-CH(NH2)-COOH
Leucine* leu L (CH3)2-CH-CH2-CH(NH2)-COOH
Methionine** met M CH3-S-(CH2)2-CH(NH2)-COOHPhenylalanine phe F Ph-CH2-CH(NH2)-COOH
Tryptophan** trp W Ph-NH-CH=C-CH2-CH(NH2)-COOH
Tyrosine tyr Y HO-p-Ph-CH2-CH(NH2)-COOH
Valine val V (CH3)2-CH-CH(NH2)-COOH
Hydrophobic: (aliphatic side chains, hydrocarbons, large bulky aromatic side groups, insoluble or less soluble; non-polar) These line the surface of mem prots within lipid bilayer
Average mol wt of a.a. is 113
**rare; *most common
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Charged amino acids
Polar no charge
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Hydrophobic amino acids
Special aa
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Peptide bond (single chemical linkage for a.a.)
From N to C terminus (carboxy gr of the 1st aa and amino gr of the 2nd)Rotation is restricted in pep bondPolyamino acids, peptide, polypeptideSize : mass in daltons (Da) or kilodaltons (kDa)R groups project from the backbone
A dalton is 1 atomic mass unit
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Three types of non-covalent bonds help proteins to fold.
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Large number of Hydrogen bonds within a polypeptide help to stabilize its three dimensional structure
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How a protein folds into a compact conformation
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Elastin molecules are cross-linked together and uncoil upon stretching
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PROTEIN STRUCTURE (4 distinct levels determine shape)
Primary; linear sequence (# and order)
Secondary; local spatial organization H bonds (random coil, -helix spiral, beta-sheet planar and turns 4 residue U shaped seg
Tertiary; 3D overall conformation of a polypeptide, hydrophobic interactions, disulfide bonds, folding of domains
Quarternary; applies to multimeric protein (2 polypep, noncovalent)
The sequence of R-groups along the chain is called the primary structure. Secondary structure refers to the local folding of the polypeptide chain. Tertiary structure is the arrangement of secondary structure elements in 3 dimensions and quaternary structure describes the arrangement of a protein's subunits.
Common regular structure; more than 60% of the protein is found to adopt these structures
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Structures
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MOTIFS are regular combinations of secondary structures specific combination with a particular topology - helix-loop-helix - zinc finger motif - coiled coil motif
DOMIANS (tertiary structures in large proteins): - fibrous / globular - much larger 100-300 a.a. (several alpha-helices and beta sheets)
- structural features or functional proline rich; SH3; Kinase domain, DNA binding domain)
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• C=O----NH (H – bonded to 4 residues away on C terminal)• 3.6 aa/turn (regular arrangement)• R- outwards (determines hydrophobic/hydrophilic character) differ on each side• proline – rare• functionally important (structural elements)• amphipathic – coiled coils, fibrous proteins
Alpha Helix
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Amphipathic Structures
-Helix
Hydophobic aa
Hydrophilic aa
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Beta-Pleated Sheet
•5-8 a.a. fully extended polypep•Planar structure•H bonds within/different polypep chain•Parallel/anti-parallel•R – project on both faces•Laterally stacked beta strands give beta sheets•Have polarity
TURNS : composed of 3 or 4 residues glycine and proline H bonds; located on prot surface
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basic zipper proteins
Helix-loop-helix / split zipper proteins
beta-beta-alpha zinc finger proteins
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•Conformation (Native state)•Key to all higher structures is the a.a. sequence•Function is dependent on its 3D structure
•Sequence homology (conserved regions): - function (homologous prots belong to same family - evolutionary relationship
•Prosthetic groups - non-covalent / covalent - e.g., zinc for metalloproteinases heme for hemoglobin
•Native state (Nascent protein undergoes folding) 8 bond angles are possible; n polypep = 8n
most stable conformation (single) native state
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Modification of Proteins: (almost all prots require this)(alter activity, life span, cellular location)
Chemical Modification:Acetylation - N terminal residue CH3CO – most prots - fatty acid acylation – membrane anchored (ras, src)Glycosylation - linear or branched CHO groups - Internal residues - many secreted and cell surface proteinsPhosphorylation - phosphate group replaces H on OH group (serine, threonine, tyrosine)
Processing:N or C terminal - pre pro insulin -procollagen - pre pro metalloproteinase (important means of keeping activity in check)
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Denaturation
- temp, pH, urea (conformation and activity are lost); disrupt noncov
- renature when removed from such condition (regain bioactivity
Shows that information for folding is contained within
ribonuclease
metalloproteinase
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• Chaperones (proteins found in bacteria and all species)
• - facilitate protein folding (molecular chaperones; chaperonins)
• large barrel shaped multimeric complex (GroEL/TCiP)
Movie
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Protein degradation:LIFE SPAN IS TIGHTLY CONTROLLED
Extracellular-Digestive system (endoproteses or exoproteases)
Intracellular-Lysosomes (membrane limited organelles)-Proteososme degrades ubiquitin targeted molecules. prot that contain the sequence (PEST) are degraded by another set of enzymes some degraded within 3 min or as long as 30 hrs
(movie)
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FORM and FUNCTION are inseparable
Pores; grooves; barrel-like structure
Protein bind other molecules (I.e., ligands for receptors on cell surface) with high degree of specificity or target molecules (substrate for enzymatic activity)
Affinity: Strength of binding (Keq; KD)
Specificity: preferential binding
Both properties depend on structural fit; complementarity
Examples: antigen : antibody (Y-shaped molecules immunoglobulins)Complementarity-determining regions at each ends
Enzyme : substrate (substrate binding site; active site)
Conformational change can be induced by substrate binding
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AntibodiesMade by B-cells of the immune system.
Multimeric proteins heavy and light chainslinked by disulfide bonds
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How noncovalent bonds mediate interactions between macromolecules
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Development of Antibodies for Cell Biology Research
Polyclonal all serum from immunized animalcontains many different antibodies to differentepitopes.
Monoclonal antibodes are produced from one plasmacell so all antibodies are identical against one epitope
Usually made in rabbits, donkeys, goats, sheep, or horse
Usually made in mouse, rat or hamster
Antibodies are secreted by activated B-cells known as plasma cells.
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Making MAb
Immunize MiceTest animal for Ab responseRemove spleenHarvest B-cellsFuse to hyridomaScreen secreted Ab for reaction to antigenExpand cell line and purify Ab.
movie
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ENZYMES:
•Catalysts @ 370C, pH 6.5 – 7.5 and aqueous•Specificity – what they bind and cleavage site•Extracellular/ Intracellular/ Tissue-specific/ House keeping•Active site – 2 important regions – bind substrate
- catalytic site•Certain a.a. side chains are important not necessarily adjacent (dependent on specific folding)
•Transition state- intermediate state
conformation change reduces activation energy
movie
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ENZYME KINETICS:
E + S E + P
Km = The Michaelis constant Affinity of the enzyme for its substrateVmax = Maximal velocity at satuarting S concentration
E + S ES EP E + P
Vmax
Km
Cons of subs [S]
Rate of productformation Km affinity [S]
Binding catalysis release
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Rx. Catalyzed by Lysozyme
1. Enzyme 1st binds the polysaccharide to form enzyme-substrate complex (ES).
2. Catalyzes cleavage of specific colavent bondForms enzyme-product complex (EP).
3. Release of product allows enzyme to act on another S.
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Feedback InhibitionA molecule other than the substrate binds to an enzymeat a special regulatory site outsidethe active site, thereby alteringthe rate at which the enzymes convertssubstrate to product.
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Membrane Proteins (A diverse group)
•Integral membrane proteins (intrinsic) embedded or transmembrane•Peripheral (extrinsic) do not interact with hydrophobic core / indirect
•Hydrophobic alpha helices in transmembrane prots•Multiple transmembrane a helices•Multiple b strands in membrane spanning barrels•Covalently attached hydrocarbons chains anchor prot to membranes
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Protein Purification and Detection:
1. Solubilization in detergents2. Centrifugation (mass or density)3. Size and charge4. Electrophoresis (charge, mass)5. Chromatography (mass, charge, binding affinity)6. Immunoblotting7. Mass Spectrometer
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Detergents
Ionic Sodium deoxycolateSodium dodecylsulfate (SDS)
Nonionic Triton X-100
Octylglucoside
hydrophilic::hydrophobic
+hydrophilic::hydrophobic
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MicellesAbove Critical Micelle Concentration (CGC)
Below CGC, No Micelles Integral proteins dissolve
MixedMicelles
detergent
phospholipid ofcell membrane
Ionic detergents bind to hydrophobic regions and core of proteins because of charge disrupts ionic and hydrogen bonds. At high conc. Completely denatures proteins.
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Centrifugation
1st step in purification of a proteinBased on differences in Mass and density
Mass= weight of sample (grams)Density= ratio of weight to volume (grams/liter)
Mass varies greatlyDensity of protein does not except for lipid or CHO additions
Differential centrifugation-separates solubleand insoluble material
Rate-Zonal-separates proteins based on theirsedimentation rate within a density gradientRate of sedimentation affected by Mass and ShapeCentrifuge too long everything into the pellet
too short no separation
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Electrophoresis
Separates proteins based on their Charge:Mass Ratio
Under applied electric field proteins move ata speed determined by their charge:mass ratio. Example twoproteins of equal mass and shape the one with the greaternet charge will move the fastest.
SDS-PAGE separates proteins based on chain length,which reflects mass, as the sole determinant of migrationrate.
Movie
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Two-Dimensional Electrophoresis1st dimension separated on charge of protein2nd dimension separated by SDS-PAGE
Charge separation is accomplished by proteinsmigrating through a pH gradient till the reach theirpI, or isoelectric point, the pH at which their netcharge is 0. This technique is isoelectric focusingIEF. After IEF strips are treated with SDS and thesecond dimension is ran.
SDS-PAGE 2-D SDS-PAGE
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Liquid Chromatography
Gel-filtration -based on polymer with pore size
Ion-exchange -based on resin with either basic or acid charge
Affinity -based on protein binding to different matrices -heparin, dyes
Antibodies -based on the affinity of Ab for protein.
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Western Blotting
SDS-PAGE Proteins transferred to membraneand antibodies are used to identify protein
movie
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Mass spectrometryLaser to fragment protein and measure peptides producedESI, MALDI, SELDI, LC-MS