Download MBP 1022, LECT 2 DAN_Oct22

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 - NH2
R
Amino acids
(monomeric subunits)
R, n=20
– determine its properties
- Diversity peptide of 4aa
has 204 possible or
160,000 sequences
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
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*
Isoleucine
Methionine**
Phenylalanine
Tyrosine
Tryptophan**
Valine
leu L
ile I
met M
phe F
tyr Y
trp W
val V
(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
Glycine
Cysteine**
gly G
cys C
NH2-CH2-COOH too flexible, fit tight spaces
HS-CH2-CH(NH2)-COOH
Sulfhyrdal group
(disulfide bond or bridge)
Proline
pro P
NH-(CH2)3-CH-COOH
Special:
Average mol wt of a.a. is 113
**rare; *most common
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
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, 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
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
Amphipathic Structures
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 = 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 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
• Chaperones (proteins found in
bacteria and all species)
• - facilitate protein folding (molecular
chaperones; chaperonins)
• large barrel shaped multimeric
complex (GroEL/TCiP)
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 (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
Antibodies
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
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
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
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
ENZYME KINETICS:
E+S
E+P
Km =
The Michaelis constant
Affinity of the enzyme for its substrate
Vmax = Maximal velocity at satuarting S concentration
Binding
E+S
catalysis
ES
release
EP
E+P
Vmax
Rate of product
formation
Km
Cons of subs [S]
Km
affinity
[S]
Rx. Catalyzed by Lysozyme
1. Enzyme 1st 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.
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.
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.
2.
3.
4.
5.
6.
7.
Solubilization in detergents
Centrifugation (mass or density)
Size and charge
Electrophoresis (charge, mass)
Chromatography (mass, charge, binding affinity)
Immunoblotting
Mass Spectrometer
Detergents
Ionic
Sodium deoxycolate
Sodium dodecylsulfate (SDS)
+hydrophilic::hydrophobic
Nonionic
Triton X-100
Octylglucoside
hydrophilic::hydrophobic
Micelles
Above Critical Micelle Concentration (CGC)
detergent
phospholipid of
cell membrane
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.
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
Electrophoresis
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
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
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 membrane
and antibodies are used to identify protein
movie
Mass spectrometry
Laser to fragment protein and measure peptides produced
ESI, MALDI, SELDI, LC-MS