2 and 3 Chem Amino Acids Protein Struc Faust CS02 03
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Transcript of 2 and 3 Chem Amino Acids Protein Struc Faust CS02 03
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Contact Information Introduction to Polypeptides
1. Central to All Aspects of Life
2. Most Abundant Macromolecules
3. Linear Polymers (20 Amino Acids)
4. Peptide Bond - covalent amide linkage
5. Polypeptide Lengths: 10,000 a.a.
6. Average Length - about 500 amino acids
7. Very High Sequence Diversity, X = 20n
Astronomical Numbers of Proteins Are Possible!!!
High sequence diversity means a high number of possibilities, X, since X = 20n,
where n is the number of amino acids in the polypeptide length, and
X is the total number of permutations available, i.e., the total amount of polypeptide sequence diversity, e.g.,
(20)2 = 400 different dipeptides (dimer),
(20)3 = 8,000 different tripeptides (trimer),
(20)6 = 64 (10)6 different hexapeptides (hexamer), and
(20)500 = 3.2 (10)600 different polypeptides (500-mers) .
Life Requires Only a Very Small
Subset of All These Possibilities
3.2 (10)600 total possibilities for 500 a.a.
Only about (10)7 are actually used in man.
So, at least (10)593 of the (10)600 are never used for the 500-mer population.
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19 Amino Acids Have an
Asymmetric* Central Carbon.
ACC ()
Tetrahedron
(pyramid)
*Glycine is NOT asymmetric.
P
D
(chiral)
P = Protonated D = Deprotonated
(see page 16)
5
Protonation and Deprotonation
-NH2
-COOH zwitterion
(Three Protonation States)
(pI)
(see page 17)
Titration Curve of Alanine
pI = 6.1 (net charge = 0)
pK* (-carboxyl) = 2.3
pK* (-amino) = 9.9
*NOTE: Best Buffering Occurs in the Flat Parts
(see page 17)
Titration of Complex Amino Acids
(Four Protonation States with an Ionizable Side Chain)
Acidic
(asp)
Basic
(lys)
pI
pI
pI
(see page 17)
-
pH = 7
14
1
[H]+ = [OH]-, (H2O, pK* = 7)
OH- ion excess
H+ ion excess, [H3O]+
Deprotonation, -H+
Protonation, +H+
*NOTE: when pH = pK, 50% P + 50% D [1:1]
SIMPLE PRINCIPLES OF PROTONATION/DEPROTONATION
more
acidic
more
basic
H2O
more
basic
more
acidic
pH = 7
14
1
[H]+ = [OH]-, (H2O, pK* = 7)
OH- ion excess
H+ ion excess
pH = pKbasic*
pH = pKacidic*
Deprotonation, -H+
Protonation , +H+
*NOTE: when pH = pK, 50% P + 50% D [1:1]
SIMPLE PRINCIPLES OF PROTONATION/DEPROTONATION
more
acidic
more
basic
Net neg.
charge
charge
loss Net pos.
charge
charge
loss
10
Two Classes of Amino Acids *
1. Hydrophilic (POLAR: loving water) - side chains (R) generally have O, N or S, viz., the 3 subgroups of basic, acidic and neutral
2. Hydrophobic (NONPOLAR: fearing water) - side chains (R) have straight, branched or cyclic carbon structures, viz., aliphatic and aromatic
*NOTE: know all 20 of the 3 letter abbreviations
Hydrophilic Amino Acids
(3 groups)
BASIC ACIDIC NEUTRAL
Arg Asp Ser Lys Glu Thr
His Asn
Gln
Cys*
Met*
*These are very weakly hydrophilic as free amino
acids, but in proteins they are usually hydrophobic.
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Hydrophobic Amino Acids
(2 groups)
Gly Phe
Ala Tyr
Val Trp Leu
Ile
Pro (aliphatic ring)
ALIPHATIC Chains AROMATIC Rings
Free*
*
not ionized
(physiological pH ~ 7.3)
(see page 19)
Protein Structure: Dictated by
Strong & Weak Chemical Bonds
15
Protein Structure: Hierarchy
of Four Basic Structural Levels
1. Primary, 1o: linear a.a. sequence (covalent bond)
2. Secondary, 2o: -helix, -sheet & -turn (H-bond)
3. Tertiary, 3o: final native 3D shape (hydrophobic)
4. Quaternary, 4o: association of multiple polypep-tides to make one protein (stabilized by all four weak, noncovalent bonds; maybe covalent ones)
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Polypeptide Formation: 1o
A. Peptide covalent bond formation
B. Peptide chain polarity
Free
terminus
Free
terminus
(N C)
* * * * *
Amide Bond
*- C
(see page 20)
Polypeptide Modifications
1. Blocking termini (covalent)
2. Disulfide bond formation (covalent)
Cystine (diamino acid)
3. Cystine occurrence (follows food digestion)
Helps to Stabilize:
2o
3o
4o
Peptide Bond is Rigid*
-
+
*(All in same plane)
H
OH
H O
H O
H
H + + -
Trans ( )
(see page 20)
Secondary Structure: 2o
1. All 2o Forms Are Stabilized by H-bonds.
2. Secondary Structure Forms Include: a. -helices (mostly right-handed) b. -pleated sheets - 2 basic types 1) Parallel
2) Antiparallel
c. -turns (hairpin turns)
20
-
Right-handed -Helix (2o)
All R groups
point away
from -helix
N
C
Polarity
Minimizes
Steric clash
R2 R1
R3
R4 R5
R6
R7
R8
Hydrogen bond
Side groups
H2N-
HOOC
Parallel -Sheet (2o)
Parallel
strands
1 2 3
N
C
Polarity
All R groups
point away
from -sheet
Polarity (see page 21)
3D Schematic of -pleated Sheet
Parallel
N
N
C
C
Parallel -Sheet (2o) (A Single Polypeptide)
N
C
-
Anti-Parallel -Sheet (2o)
Anti-parallel
strands
1 2
3 Polarity
N
C
Polarity
All R groups
point away
from -sheet
(see page 21)
25
3D Schematic of -pleated Sheet
Anti-parallel
N
N
C
C
Anti-Parallel -Sheet (2o) (A Single Polypeptide)
C
N
-Turn (2o)
.
.
.
. . .
.
.
. .
.
. .
.
.
H-bond
(R)
-
Tertiary Structure (3o):
the Native 3D shape
1. Hydrophobic interactions are the principal stabilizing force of tertiary structure.
2. Core is mainly hydrophobic side chains.
3. Domains are discreetly packed elements that result from hydrophobic side chain packing
and assembling of all secondary structures.
4. Tertiary structures may have 1 domain or more.
Tertiary Structure: One Domain
(basic immunoglobulin domain)
-S-S-
30
Two Domains (or more, 3o) Bence Jones proteins: Multiple myeloma
-S-S-
NH2
HOOC
Quaternary Structure (4o):
Multimeric Proteins (Hb)
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Polypeptide Folding Events
1. Peptide bond formation occurs first (1o). 2. Primary structure contains folding instructions. 3. Chaperones can provide folding assistance. 4. Chaperones can also correct some misfolding. 5. But polypeptides are
degraded back to simple amino acids in
organelle structures called proteosomes.
Protein Denaturation
1. Loss of ALL higher ordered structures,
i.e., complete unfolding of 2o, 3o & 4o,
producing a polypeptide random coil.
2. Various types of denaturants available:
a. Heat () b. Detergents & organic solvents c. Strong acids and bases d. 8M urea and 6M guanidine HCl e. Heavy metals, e.g., Pd, Cd, Hg, etc.
Protein Modifications
1. Generally occur posttranslationally. 2. Over 500 different kinds are known. 3. Some more common ones include:
a. Disulfide/sulfhydryl formation
b. Phosphorylation (some regulatory)
c. Glycosylation (O-linked & N-linked)
d. Vitamin additions (co-factors).
35
Phosphorylation
Phosphates joined
to hydroxyl side
chains (-OH), i.e.,
1. Ser
2. Thr
3. Tyr
-
Glycosylation ( )
Sugars joined
to (-OH) side
chains, i.e.,
1. Ser
2. Thr
Sugar
Glycosylation (N-linked)
Sugar joined to
Asn side chain
(-NH2) only.
Sugar
Context:
1. Asn-X-Ser 2. Asn-X-Thr
X = Pro
Biotin (Vitamin H) Addition
Side Chain (-NH2)
Lys only
Biotin: a
prosthetic
group (1)
Apoprotein (2)
Holoprotein = (1 + 2)
Disulfide Bond Reactivity
irreversible
reversible
(see page 23)
40
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Protein Purification and
Characterization Methods
1. Proteins Absorb in the UV (@210 or 280 nm)
2. Protein Solubility: surface side chains
3. Dialysis: small contaminant removal
4. Electrophoresis of Proteins
a. Separation by charge
b. Separation by size
5. Structural characterizations: 1o, 2o, 3o, 4o
UV Absorption Spectra
peptide bond (210 nm)
trp
tyr
phe
(see page 24)
(bases)
Protein Solubility
Distilled water: leads to mild attraction for some proteins
+
(see page 24)
Protein Solubility
Physiological or moderate salt (salting in):
leads to weak repulsion of proteins
Salting out: protein precipitation at high salt concentrations
(salt ties up water, limiting protein solubility)
(see page 24)
-
Protein Solubility
Varies with different pHs
(Excess H+) (Excess OH-)
(Net charge = 0)
< pI > pI
pI
OH -
H +
Net = 0
-
+ (see page 24)
45
Dialysis: Small molecule removal
time
Cutoff:
10 kDa
(see page 25)
Electrophoresis: Separation by Charge
time
(1-)
(3-)
(4-)
(2-)
Protein
migration
Protein
Net Charge
(see page 25)
Electrophoresis: Separation by Size (Denaturing SDS PAGE)
time
25 kDa
50 kDa
100 kDa
Largest
Smallest
Start Finish
Sample: + SDS
(see page 25)
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Protein Structure Determination
Primary: amino acid composition, peptide
fragmentation, Edman degradation (for the
sequence), and mass spectrometry (seq.)
Secondary: CD and ORD (spectroscopies)
Tertiary: X-ray diffraction and NMR
Quaternary: X-ray diffraction, NMR & others
1o Structure Characterization:
Overlapping Peptide Mapping*
*Edman degradation: yields pepide sequence (~50 aa, NC)
*
*
*
N C
50
Neurodegenerative Disorders (Diseases of malfolded proteins)
1. Human Prion Diseases (amyloids: fibrous tangles)
a. Bovine spongiform encephalopathy (BSE)
b. Sporadic Creutzfeld-Jakob disease (1/106)
c. Gerstmann-Straussler-Scheinker dis. (1/107)
d. Fatal familial insomnia (1/107)
e. Alpers syndrome: progressive infantile poliodystrophy
f. Kuru (New Guinea tribe ancestor ritual, C. Gadjusek, 76)
2. Alzheimers: aggregated protein (unprocessed?)
3. Parkinsons: aggregated protein (unprocessed?)
Human Prions
1. Infectious protein: agent of all prion diseases
2. Polypeptide: ~250 amino acids (S. Prusiner, 97)
3. Only protein is infectious, i.e., no nucleic acid.
4. Native form exists on nerve cell surfaces.
5. Normal function remains unknown.
6. Conformational change believed responsible for etiology of the disease condition.
-
Prion Structure & Properties
1. Normal prion protein: 3 -helices 2. Abnormal protein: -sheet increases > 40% 3. Abnormal: resistant to proteolytic digestion
4. Abnormal: induces conversion of metastable
normal prions to stable abnormal prions
5. Amyloid: neurotoxic fibrillar deposits
6. Brain cell death ensues (apoptosis?)
7. Prognosis: eventually the patient dies.
Prion Conversion Mechanism: PrPC PrPSc
Normal Prion
Abnormal Prion (Long term process)
Normal Type Disease Type
PrPC PrPSc
-Helix to -Sheet Conversion
55
Proposed Model of Prion
Amyloid Fibril Formation
-
Prions: molecular genetics
met/val asp (GAC/T)
129* 178
asn (AAC/T)
CJD FFI
wt-1o
human
~250 a.a.
mutation
*met and val variants exist in general population
Normal Human vs. vCJD Brain
N vCJD (H&E) (H&E)
Mouse Knock In Model: CJD
(Human Diseased Prion Gene)
H & E mAb HuP Mouse Brain Sections
Alzheimers Disease
60