CHAPTER 3 November 1

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CHAPTER 3- November 1 Proteins HIGHLIGHTS
Structure and Chemistry of amino acids Linkage
to form a polypeptide monomer to polymer Forces
that guide folding Modifications and
degradation Functional design Common
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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• 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 polymerpeptide or
  polypeptide

COOH Amino acids
(monomeric subunits) H - C - NH2 R,
n20 determine its properties
R - Diversity peptide of 4aa has
204 possible or 160,000 sequences
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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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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
Alanine ala A CH3-CH(NH2)-COOH
 
 
Leucine leu L (CH3)2-CH-CH2-CH(NH2)-COOH
 
Isoleucine ile I CH3-CH2-CH(CH3)-CH(NH2)-COOH
 
Methionine met M CH3-S-(CH2)2-CH(NH2)-COO
H
Phenylalanine phe F Ph-CH2-CH(NH2)-COOH
Tyrosine tyr Y HO-p-Ph-CH2-CH(NH2)-COOH
Tryptophan trp W Ph-NH-CHC-CH2-CH(NH2)-COO
H
 
Valine val V (CH3)2-CH-CH(NH2)-COOH

 
Special
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
 
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 bond Polyamino acids, peptide,
polypeptide Size 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, a-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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Alpha Helix

• CO----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

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Amphipathic Structures
a-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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beta-beta-alpha zinc finger proteins
Helix-loop-helix / split zipper proteins
basic zipper 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 Acetyla
tion - 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 proteins Phosphorylation -
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) Complementari
ty-determining regions at each ends Enzyme
substrate (substrate binding site active
site) Conformational change can be induced by
substrate binding
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Antibodies
Made by B-cells of the immune system.
Multimeric proteins heavy and light chains linked
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
Antibodies are secreted by activated B-cells
known as plasma cells.
Polyclonal all serum from immunized
animal contains many different antibodies to
different epitopes.
Usually made in rabbits, donkeys, goats, sheep,
or horse
Monoclonal antibodes are produced from one
plasma cell so all antibodies are identical
against one epitope
Usually made in mouse, rat or hamster
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Making MAb
Immunize Mice Test animal for Ab response Remove
spleen Harvest B-cells Fuse to hyridoma Screen
secreted Ab for reaction to antigen Expand cell
line and purify Ab.
movie
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• ENZYMES
• Catalysts _at_ 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 substrate Vmax Maximal velocity at
satuarting S concentration E S ES EP E P
Binding catalysis release
Vmax
Rate of product formation
Km affinity S
Km
Cons of subs S
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Rx. Catalyzed by Lysozyme

• Enzyme 1st binds the polysaccharide to form
• enzyme-substrate complex (ES).
• Catalyzes cleavage of specific colavent bond
• Forms enzyme-product complex (EP).
• Release of product allows enzyme to act on
  another S.

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Feedback Inhibition
A molecule other than the substrate binds to an
enzyme at a special regulatory site outside the
active site, thereby altering the rate at which
the enzymes converts substrate 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
• Solubilization in detergents
• Centrifugation (mass or density)
• Size and charge
• Electrophoresis (charge, mass)
• Chromatography (mass, charge, binding affinity)
• Immunoblotting
• Mass Spectrometer

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Detergents
Ionic
Sodium deoxycolate
Sodium dodecylsulfate (SDS)
Nonionic
Triton X-100
Octylglucoside
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Micelles
Above Critical Micelle Concentration (CGC)
Mixed Micelles
Below CGC, No Micelles Integral proteins dissolve
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 protein Based on
differences in Mass and density Mass weight of
sample (grams) Density ratio of weight to
volume (grams/liter) Mass varies greatly Density
of protein does not except for lipid or CHO
additions
Differential centrifugation-separates soluble and
insoluble material
Rate-Zonal-separates proteins based on
their sedimentation rate within a density
gradient Rate of sedimentation affected by Mass
and Shape Centrifuge too long everything into
the pellet too short no separation
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Electrophoresis
Separates proteins based on their ChargeMass
Ratio
Under applied electric field proteins move ata
speed determined by their chargemass ratio.
Example two proteins of equal mass and shape the
one with the greater net charge will move the
fastest.
SDS-PAGE separates proteins based on chain
length, which reflects mass, as the sole
determinant of migration rate.
Movie
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Two-Dimensional Electrophoresis
1st dimension separated on charge of protein 2nd
dimension separated by SDS-PAGE
Charge separation is accomplished by
proteins migrating through a pH gradient till the
reach their pI, or isoelectric point, the pH at
which their net charge is 0. This technique is
isoelectric focusing IEF. After IEF strips are
treated with SDS and the second 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 membrane and
antibodies are used to identify protein
movie
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Mass spectrometry
Laser to fragment protein and measure peptides
produced ESI, MALDI, SELDI, LC-MS