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

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

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
COOH
H - C - NH
2
R
Amino acids
(monomeric subunits)
R, n=20
– determine its properties
- Diversity peptide of 4aa has 20 4 possible or
160,000 sequences

R Chains (Special Properties)
• Hydrophilic (surface)
- Basic +ve lys(K), arg(R), (His)
- Acidic -ve
- polar glu(E), asp(D)
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

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
Leucine*
Isoleucine
Methionine**
Phenylalanine
Tyrosine
Tryptophan**
Valine ala A leu L ile I met M phe F tyr Y trp W val V
CH3-CH(NH2)-COOH
(CH3)2-CH-CH2-CH(NH2)-COOH
CH3-CH2-CH(CH3)-CH(NH2)-COOH
CH3-S-(CH2)2-CH(NH2)-COOH
Ph-CH2-CH(NH2)-COOH
HO-p-Ph-CH2-CH(NH2)-COOH
Ph-NH-CH=C-CH2-CH(NH2)-COOH
(CH3)2-CH-CH(NH2)-COOH
Special:
Glycine
Cysteine**
Proline gly G cys C pro P
Average mol wt of a.a. is 113
**rare; *most common
NH2-CH2-COOH too flexible, fit tight spaces
HS-CH2-CH(NH2)-COOH Sulfhyrdal group
(disulfide bond or bridge)
NH-(CH2)3-CH-COOH kink, cyclic ring, rigid

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 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

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

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, 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

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)

Alpha Helix
•
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

a
-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

beta-beta-alpha zinc finger proteins
Helix-loop-helix / split zipper proteins basic zipper 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 = 8 n 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 CH
3
CO – 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)

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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Movie

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)

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 (K eq
; K
D
)
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

Made by B-cells of the immune system.
Multimeric proteins heavy and light chains linked by disulfide bonds

How noncovalent bonds mediate interactions between macromolecules

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

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

ENZYMES:
• Catalysts @ 37 0 C, 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

ENZYME KINETICS:
E + S E + P
K m
= The Michaelis constant
Affinity of the enzyme for its substrate
V max
= Maximal velocity at satuarting S concentration release
E + S
Binding
ES catalysis
EP E + P
V max
Rate of product formation
K m
Cons of subs [S]
Km affinity [S]

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

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.

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 detergents
2. Centrifugation (mass or density)
3. Size and charge
4. Electrophoresis (charge, mass)
5. Chromatography (mass, charge, binding affinity)
6. Immunoblotting
7. Mass Spectrometer

Ionic Sodium deoxycolate
Sodium dodecylsulfate (SDS)
+hydrophilic ::hydrophobic
Nonionic Triton X-100
Octylglucoside hydrophilic ::hydrophobic

Above Critical Micelle Concentration (CGC) detergent phospholipid of cell membrane
Below CGC, No Micelles Integral proteins dissolve
Mixed
Micelles
Ionic detergents bind to hydrophobic regions and core of proteins because of charge disrupts ionic and hydrogen bonds. At high conc. Completely denatures proteins.

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

Separates proteins based on their Charge:Mass Ratio
Under applied electric field proteins move ata speed determined by their charge:mass 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

1 st dimension separated on charge of protein
2 nd 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

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.

SDS-PAGE Proteins transferred to membrane and antibodies are used to identify protein movie

Laser to fragment protein and measure peptides produced
ESI, MALDI, SELDI, LC-MS