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

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

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

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

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

Charged amino acids

Polar no charge

Hydrophobic amino acids

Special aa

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

Three types of non-covalent bonds help proteins to fold.

Large number of Hydrogen bonds within a polypeptide help to stabilize its three dimensional structure

How a protein folds into a compact conformation

Elastin molecules are cross-linked together and uncoil upon stretching

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

Structures

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)

• 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

Amphipathic Structures

-Helix

Hydophobic aa

Hydrophilic aa

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

basic zipper proteins

Helix-loop-helix / split zipper proteins

beta-beta-alpha zinc finger proteins

•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

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)

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

• Chaperones (proteins found in bacteria and all species)

• - facilitate protein folding (molecular chaperones; chaperonins)

• large barrel shaped multimeric complex (GroEL/TCiP)

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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

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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

AntibodiesMade by B-cells of the immune system.

Multimeric proteins heavy and light chainslinked by disulfide bonds

How noncovalent bonds mediate interactions between macromolecules

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.

Making MAb

Immunize MiceTest animal for Ab responseRemove spleenHarvest B-cellsFuse to hyridomaScreen secreted Ab for reaction to antigenExpand cell line and purify Ab.

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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

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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

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.

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.

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

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

Detergents

Ionic Sodium deoxycolateSodium dodecylsulfate (SDS)

Nonionic Triton X-100

Octylglucoside

hydrophilic::hydrophobic

+hydrophilic::hydrophobic

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.

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

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.

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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

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.

Western Blotting

SDS-PAGE Proteins transferred to membraneand antibodies are used to identify protein

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Mass spectrometryLaser to fragment protein and measure peptides producedESI, MALDI, SELDI, LC-MS