PEPTIDE STRUCTURE - FUNCTION

Rational Design of Peptides - Driving ForceRational Design of Peptides - Driving Force

SEQUENCE STRUCTURE

CRYSTALLOGRAPHYNMR – HIGH RES

ABS, FLUORES,CD, IR - LOW

SEQUENCING

H2NCHC OH

O

R

HHNCHC OH

O

R

+

H2NCHC

O

R1

HNCHC OH

O

R2

PEPTIDE BOND FORMATIONPEPTIDE BOND FORMATIONAA1 AA2

H20

DIPEPTIDE

PROTEASES

PHYSICO-CHEMICAL PROPERTIES

PHYSICAL PROPERTIES ADDITIVE

LEGNTH, MASS

NOT ADDI IVE Pka => AA1+AA2 ===== DIPEPEPTIDE

ENERGETICS, REACTIVITY ETC

STRUCTURE OF THE PEPTIDE BOND

GEOMETRICAL CONSTRAINTS - CONFORMATIONS

ALLOWED NOT-ALLOWED ANGLES

DIPOLE ORIGIN OF PEPTIDE BOND PLANE

NOT ALL CONFORMATIONS POSSIBLEPREFERED CONFORMATIONS

STRUCTURAL MOTIFS - FUNCTIONAL

1. HELIX - -HELIX, 3-10 HELIX

2. -SHEETS (Parallel, Anti-Parallel

3. -TURNS

4. RANDOM COILS

helix• α-helix (30-35%)

– Hydrogen bond between C=O (carbonyl) & NH (amine) groups within strand (4 positions apart)

– 3.6 residues / turn, 1.5 Å rise / residue– Typically right hand turn– Most abundant secondary structure– α-helix formers: A,R,L,M,E,Q,K

the alpha-helix: repeating i,i+4 h-bonds

2

1

3

4

5

7

8

9

6

10

11

12 right-handed helical region of phi-psi space

hydrogen bond

The -helix, with i,i+4 h-bonds, is not the only way to have local hydrogen bonding of the backbone to itself.

The 310 helix has hydrogen bonds between residues i and i+3

The helix has hydrogen bonds between residues i and i+5.

For a number of reasons almost all helices in proteins are -helices--include backbone, side chain steric issues, van der Waals contacts, H-bondgeometry

-helix 310 helix helix

these are poly-Ala,so the gray balls on theoutside are -carbons from the side chains

sheet & turn• β-sheet / β-strand (20-25%)

– Hydrogen bond between groups across strands– Forms parallel and antiparallel pleated sheets– Amino acids less compact – 3.5 Å between adjacent

residues– Residues alternate above and

below β-sheet– β-sheet formers: V,I,P,T,W

β-turnShort turn (4 residues)Hydrogen bond between C=O &

NH groups within strand

(3 positions apart)Usually polar, found near surfaceβ-turn formers: S,D,N,P,R

TURNS

Others

• Loop (bridging region)– Regions between α-helices and β-sheets– On the surface, vary in length and 3D

configurations– Do not have regular periodic structures– Loop formers: small polar residues

• Coil (40-50%)– Generally speaking, anything besides α-helix, β-

sheet, β-turn

Principal types of secondary structure found in proteins

Repeating () values

-63o -42o

-57o -30o

-119o +113o

-139o +135o

-helix(15) (right-handed)

3helix(14)

Parallel -sheet

Antiparallel -sheet

STRUCTURES IN ACTION

GCN4 “leucine zipper” (green) bound asa dimer (two copies of the polypeptide) to target DNA

The GCN4 dimer is formed throughhydrophobic interactions betweenleucines (red) in the two polypeptide chains

Leu

Leu

TECHNIQUES – PEPTIDE COMFORMATIONCD X-ray Crystallography

NMR

Do Small Peptides have Conformation

Yes & No. S-Peptide Ribonuclease A – Helical structure in solution

Use of Helix Inducing Solvents – TFE and N-Propanol

Helix Induction and Propensity

CONFORMATIONAL TRANSITION

PROPENSITY CALCULATION

BIOLOGY OF PEPTIDES

RIBOSOMALPROTEINS NON-RIBOSOMAL

PEPTIDESSPECIFIC ENZYMES

PROTEOLYTICALYPROCESSED

DEGRADED TO AA

(Antibiotics, phytochelatins)

(Enzymes)MHC Peptides

CHEMICAL METHOD –PEPTIDE SYNTHESISCHEMICAL METHOD –PEPTIDE SYNTHESIS

STAGE 1: ASSEMBLE AA ON POLYMER SUPPORT (R – PROTECTED) NON-REACTIVE

STAGE 2: CLEAVE THE SYNTHESIZED PEPTIDE a) CLEAVAGE OF CHAIN b) DE-PROTECT SIDE CHAIN

STAGE 3: PURIFY CRUDE PEPTIDES – HPLC

STAGE 4: STORAGE – LYOPHILIZE, SPEEDVAC, ETC

STAGE 5: SEQUENCE, MALDI-TOF

SOLID-PHASE PEPTIDE SYNTHESIS (SPPS)SOLID-PHASE PEPTIDE SYNTHESIS (SPPS)

STAGE 1: a) Attach N-terminal + Side Chain Protected to Polymer Support (Activation of C & Coupling to Support) b) Deprotection (N-term )

c) Coupling Next AA (Protected) d) Deprotection (N-term) Continued ….

V8 Protease

“Conformational Trap”

LYGSTSQE VASVKQAFDAVGVK

NH-VASVKQAFDAVGVK-OH NH-LYGSTSQE-OH

“Proteolysis”

“Reverse Proteolysis”

LYGSTSQE VASVKQAFDAVGVK

Protease-mediated Protein Splicing – Nature’s Choice

TAAAKFE

“Conformational Trap” can act alone

Conformational Trap of product – ambient conditions, easyIsolation, and Purification of Products

Applications

1. Ability to Incorporate Non-Natural aminoacids or synthesizeMan-made peptides or proteins of therapeutic interest

Semisynthetic Insulin, Hemoglobin, and IL-10

Sortases – Glycoprotein synthesis

Laboratory reagents :- Protein with reporter groups, Kinases orPhosphotases with Pmp(phosphonomethylene phenylalanine)