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.

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.

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)

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)

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

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)

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