Peptide+structure
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Transcript of Peptide+structure
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)