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47
Peptide and Protein AnalysisPrimary (1°) structure of a peptide or protein is the amino acid sequence
Amino acid analyzer- automated instrument to determine the amino acid content of a peptide or protein. Individual amino acids areseparated by hplc, then detected by post-column derivatization
1972 Nobel Prize in ChemistryWilliam Stein Stanford Moore
peptide-or-
protein
[H] reduce anydisulfidebonds
Enzymaticdigestion
R CO2
NH3 individualamino acids-or-
H3O+, Δ
liquidchromatography
derivatize w/ninhydrin
Detected w/UV-vis
Different amino acids have different chromatographicmobilities (retention times)
48
Reaction of primary amines with ninhydrin
Intense purple color
So, why is it necessary to use a post- rather than pre-column derivatization protocol?
Why are there are only 17 AA’s in the chromatogram?
Amino Acid Analysis Chromatogram
O
O
N
O
O
R CO2
NH3
O
O
O+
25
49
Fluorescence Detection- less background, greater sensitivity,lower detection limits
Absorption spectroscopy- wavelength that light absorbs, moloeculesare in an electronically excited state
Emission spectroscopy- the excited molecules relax by emissionof a photon.
Fluorescence- excitation wavelength and emission wavelength are different. Molecule will emit light at longer (lower energy)wavelength than is absorbs.
50
Fluorescent tags Dansyl- detected by UV or fluorescence
R CO2
NCH3H3C
SO O
Cl
+
NH3C CH3
S OO
NH
R CO2
NH3
Dansyl chloride
OPA (o-phthalaldehyde)- detected by fluorescence
R CO2
NH3 +CHO
CHO
SHHO
N
S
R
CO2
OH
highly fluorescent
26
51
Reversed-phase (C-18) HPLC Trace5 pmols amino acids w/ OPA, HOCH2CH2SH
52
Attomol detection w/ laser induced fluorescence
10-3 milli10-6 micro10-9 nano10-12 pico10-15 fempto10-18 atto10-21 zepto Avagadro’s number 1023
N CHO
O
CO2
N
N
R
CO2
S
OH
CO2
excitation: 488 nm
emission: 560 nm
27
53
Peptide and Protein Sequences: primary (1°) structure- amino acid sequence
N-labeling with Sanger’s reagent: Sanger’s (2,4-dinitrofluorobenzene)reagent reacts with the N-terminal amino group and has a diagnostic UV absorbance that is detected after enzymatic digestion and amino acid analysis
NO2
O2N
F
H3N
O
R1 HN CO2+
!
NH
O
R1 HN CO2nucleophilic
aromaticsubstitution
NO2
O2N
NH
O
R1
NH2
NO2
O2N
+ plus other unlabeled amino acids
enzymatic
digestion
-or-
H3O+, !
N-terminal amino acid is specifically labeled with a unique UV chromophore
54
C-terminal sequencing:Carboxypeptidase- enzyme that hydrolyzed amide bonds of a peptide
or protein starting from the C-termial end (exopeptidase)
NH
O
HN
R2
O
NH
R1
O
O
R3O
H3N NH
O
HN
R2
OR3O
H3N O +Carboxypeptidase
Zn2+, H2OHN
R1
O
O
derivatize and identify by HPLC
peptide has a new C-terminal AA
Hydrolyze peptide with hydazine (H2N-NH2)
NH
O
HN
R2
O
NH
R1
O
O
R3O
H3N
H2NNH2
HN
Rn
O
NHNH2HN
R1
O
O +
C-terminal AAis still an amino acid
All other AA's areconverted to thehydrazides
28
55
Edman Degradation: chemical method for the sequential cleavage andidentification of the amino acids of a peptide, one at a time starting from the N-terminus. Reagent: Ph-N=C=S, phenylisothiocyanate)
H2N CO2
S
C
NPh
+ H2N
O
R1 HN CO2
pH 9.0
then H+ HN
N OS
R1
Ph
+
N-phenylthiohydantoin:separated by HPLC, detected by UV-vis
-1 peptide with a new N-terminal amino acid (repeat degradation cycle)
56
Fluorescent Edman sequencing reagent
O
CO2H
OHO
Fluorescein(a common fluorescent dye)
O
CO2H
OHO
NC
S
Fluorescein Isothiocyanate(a fluorescent Edman reagent)
Peptide sequencing by Edman degradation: Monitor the appearance of N-phenylthiohydantoin over time to get the peptide sequence. Good for peptides up to ~ 25 amino acids long. Longer peptides andproteins must be cut into smaller fragments before Edman sequencing
29
57
Peptide sequencing by tandem mass spectrometryIonization: SIMS (secondary ion mass spectrometry)Time-of-flight (TOF) mass spectrometer
Methods to get large, polar molecules into the gas phase for MS analysis
FAB: Fast Atom BombardmentMALDI: Matrix-Assisted Laser Desorption IonizationESI: Electrospray Ionization
Mass spectrometry gives mass/charge (m/z) ratio
“Introduction to Proteimics: Tools for the New Biology,” Liebler, D. C., Humana Press: 2002
58
Mass spectrometry is a gas phase technique. Peptides (and proteins) are charged, polar, high molecular weight molecules (ions). How can peptides and proteins becoaxed into the gas phase?
Electrospray ionization (ESI): analyte is introduced into the massspectrometer as an aerosol.
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
- +
+
to the mass analyzer
liquid chromatographyor capillary electrophoresis(separate the analytes)
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+ +++++
- +
+ ++++++
+++
++++
+++++
++++
+++
++++
+++++
+
+ + ++++
- +
+++++
++
+++
++++
+++++
++++
+++
++++
+++++
+
+ +++++
- +
+ ++++++
+++
++++
+++++
++++
+++
++++
+++++
+
+ +++ ++
- +
+ ++++++
+++
++++
+++++
++++
+++
++++
+++++
+
+ +++ ++
+
+
++
+
- +
+ ++
Coulombicfission
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
+
+
++
+
- +
++
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
- +
+
+
+
+
+
++
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
- +
+
+
+
+
+
+
30
59
MALDI ionization (matrix-assisted laser desorption): analyte isco-crystallized with an organic molecule that has anintense UV absorption. A laser that is tuned to the absorption of the matrix, is “pulsed” at the MALDImatrix and energy is indirectly transferred to the analyte.
++
+
+
+
+
to the mass analyzer
Laser pulse
++
+
+
+
+
+
+
2002 Nobel Prize in ChemistryJohn Fenn (ESI)Koichi Tanaka (MALDI)
60
[M + H]+ acceleratedinto MS
collisioncell
(He, Ar, Xe)
analyzefragments
to get sequence
Collision of the [M+H]+ ion with the gas causes it to fragment, analysis of these fragments ions gives sequence information
Many peptides and proteins give multiply charged ions
CID: collision induced dissociation
OCCH
R
H2N
N-terminalfragment
CO2HCH
R
H3N
C-terminalfragment
H2N CH C
R1
OHN CH C
R2
OHN CH C
R3
OHN CH C
R4
OH
O
a1 b1 c1
x1 y1 z1
a2 b2 c2
x2 y2 z2
a3 b3 c3
x3 y3 z3
charge to C-terminus
charge to N-terminus
31
61
Peptide sequencing by tandem mass spectrometry
to thedetector
Q1ElectrosprayIon Source Q3Collision Cell (Q2)Nanospray
Capillary
Select peptide to be analyzed
fragment thepeptide
Analyze thepeptide fragments
1000 1500 2000 2500 3000 m/z
1116
.67
1287
.73
1375
.76
1424
.85 1505
.77
1665
.89
1811
.85
2005
.07
2476
.21
2550
.52
2719
.48
1849
.12
1574
.20
1247
.70
Peptides fragment ina predictable manner
H2N CH C
R1
OHN CH C
R2
OHN CH C
R3
OHN CH C
R4
OH
O
b1
y1
b2
y2 y3charge to
C-terminus
charge to N-terminus
b3
Select m/z 1505.8 for Q2
62
average exact - HN-CHR-COGlycine G 75.07 75.03 57.1Alanine A 89.10 89.05 71.1Serine S 105.09 105.04 87.1Proline P 115.13 115.05 97.1Valine V 117.15 117.08 99.1Threonine T 119.12 119.06 101.1Cysteine C 121.16 121.02 103.1Isoleucine I 131.18 131.09 113.2Leucine L 131.18 131.09 113.2Asparagine N 132.12 132.05 114.1Aspartic Acid D 133.11 133.04 115.1Glutamine Q 146.15 146.07 128.2Lysine K 146.19 146.11 128.1Glutamic Acid E 147.13 147.13 129.1Methionine M 149.21 149.05 131.2Histidine H 155.16 155.02 137.1Phenylalanine F 165.19 165.19 147.2Arginine R 174.20 174.11 156.2Tyrosine Y 181.19 181.07 163.2Tryptophan W 204.23 204.09 186.2
Amino Acids Sorted by Mass
H2N CH C
R1
OHN CH C
R2
OHN CH C
R3
OHN CH C
R4
OH
O
b1
y1
b2
y2 y3
b3
32
63
Some ambiguities with MS sequencingleucine (L) vs isoleucine (I): difficult to distinguish, must look
at fragmentation of the sidechain
lysine (K, m/z=128.09) vs glutamine (Q, m/z = 128.06)
gly (G) + gly (G) = 114.04 = asn (N)= 114.04ala (A) + gly (G) = 128.06 = gln (Q) = 128.06 = lys (K)= 128.09gly (G) + val (V) = 156.09 = arg (R) = 156.10ala (A) + asp (B) = glu (Z) + gly (G) = 186.06 = trp (N) = 186.08ser (S) + val (V) = 186.1 = trp (N) = 186.08
CO2
NH3
CO2
NH3
CO2
NH3
CO2
NH3
H2N
O
H2N Ac2O
Ac2O
CO2
NH3
HN
O+ 42 amu's
no reaction
64
H2N CH C
CH2
O
CH2
C
OH
O
HN CH C
CH2
O
HC CH3
CH3
HN CH C
HC
O
CH3
CH3
HN CH C
HC
O
CH3
CH2
CH3
NH
CH C
CH2
O
OH
HN CH C
CH2
O
HC CH3
CH3
HN CH C
HC
O
CH3
CH2
CH3
HN CH C
HC
O
CH3
CH3
HN CH C
CH2
O
CH2
C
OH
O
HN CH C
CH2
O
OH
HN CHC
CH2
OH
O
CH2
CH2
CH2
NH2
H2N CH C
CH2
O
CH2
C
OH
O
HN CH C
CH2
O
HC CH3
CH3
HN CH C
HC
O
CH3
CH3
HN CH C
HC
O
CH3
CH2
CH3
HN CH C
CH2
O
OH
b5 fragment: m/z = 542.1
H3N CH C
CH2
O
HC CH3
CH3
HN CH C
HC
O
CH3
CH2
CH3
HN CH C
HC
O
CH3
CH3
HN CH C
CH2
O
CH2
C
OH
O
HN CH C
CH2
O
OH
HN CHC
CH2
OH
O
CH2
CH2
CH2
NH2y6 fragment: m/z = 688.3
-or-
CID
H+
33
65
H2N CH C
CH2
O
CH2
C
OH
O
HN CH C
CH2
O
HC CH3
CH3
HN CH C
HC
O
CH3
CH3
HN CH C
HC
O
CH3
CH2
CH3
NH
CH C
CH2
O
OH
HN CH C
CH2
O
HC CH3
CH3
HN CH C
HC
O
CH3
CH2
CH3
HN CH C
HC
O
CH3
CH3
HN CH C
CH2
O
CH2
C
OH
O
HN CH C
CH2
O
OH
HN CHC
CH2
OH
O
CH2
CH2
CH2
NH2
b1
y10
b2
y9
b3
y8
b4
y7
b5
y6
b6
y5
b7
y4
b8
y3
b9
y2
b10
y1
Glu Leu Val Ile Ser Leu Ile Val Glu Ser Lys
129 113 99 113 87 113 113 99 129 87 145
66
34
67
Enzymatic and chemical cleavage of peptides and proteins atdefined sites
Enzymatic• trypsin: cleaves at the C-terminal side of basic residues,
Arg, Lys but not His
• chymotrypsin: cleaves at the C-terminal side of aromatic residuesPhe, Tyr, Trp
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
NH3
NH
O
HN
O
H3N
R3
O
HN
R1O
H3NCO2
NH3
O +trypsin
H2O
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2 N
HO
HN
O
H3N
R3
O
HN
R1O
H3NCO2
O +chymotrypsin
H2O
68
• thermolysin: cleaves at the N-terminal side of hydrophobic residuesPhe, Trp, Leu
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2 +thermolysin
H2O
NH
O
H3N
O
NH
R3
O
HN
R1O
H3NCO2
O
Chymotrypsin cleavage products Trypsin cleavage products
Tyr ArgAsp-Asn-Gln Leu-LysGly-Gly-Phe Ile-Arg-Pro-LysLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp Tyr-Gly-Gly-Phe-Leu-Arg
Trp-Asp-Asn-Gln
Trypsin:
Chymotrypsin:Trp-Asp-Asn-Gln
Asp-Asn-GlnLeu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp
Leu-Lys Ile-Arg-Pro-Lys Arg Tyr-Gly-Gly-Phe-Leu-Arg
Gly-Gly-Phe Tyr
35
69
Chemical cleavage of peptides and proteins at defined sites
• Cyanogen bromide (Br-CN): cleaves to the C-terminal side of methionine residues
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
SH3C
+NH
O
H3N
R3
O
HN
R1O
H3N
CO2
HN
Br-CN
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
SH3C CN
O
NH
R3
O
HN CO2
NH
O
R1O
H3N
- H3CSCN
OH2
O
NH
R3
O
HN CO2
NH
O
R1O
H3N O
H
H+
NH
NH
O
O
NH
O
R1O
H3N
HN CO2
OH
C-terminal
homo-serine
H2O
70
• cleavage at N-terminal side of cysteine residues
Cys is converted to Ser
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
SH
+
(H3CO)3PO
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
S
CH3
- H3CSCN
H+
H3O
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
S
CH3
CN
Br-CN
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
O2H
NH
O
HN
O
NH
R3
O
HN
R1O
H3NCO2
OH
NH
H2N
O
HN
R3
O
NH
R1O
H3N CO2
O
O
NH
O
HN
R3
O
NH
R1O
H3N CO2
O
H3NO
HO
36
71
EPIDERMAL GROWTH FACTOR (EGF)
H2N-ASN1•SER2•TYR3•PRO4•GLY5•CYS6•PRO7•SER8•SER9•TYR10•ASP11•GLY12•TYR13•CYS14•LEU15•ASN16•GLY17•GLY18•VAL19•CYS20•MET21•HIS22•ILE23•GLU24•SER25•LEU26•ASP27•SER28•TYR29•THR30•CYS31•ASN32•CYS33•VAL34•ILE35•GLY36•TYR37•SER38•GLY39•ASP40•ARG41•CYS42•GLN43•THR44•ARG45•ASP46•LEU47•ARG48•TRP49•TRP50•GLU51•LEU52•ARG53-CO2H
Trypsin Chymotrypsin Cyanogen Bromide
Disulfides bridges at: Cys6 - Cys20Cys14 - Cys31Cys33 - Cys42
S. Cohen et al. J. Biol. Chem. 1972, 247, 5928-5934 1972, 247, 7612-7621 1973, 248, 7669-7672
72
DNA mRNA protein
37
73
Protein and Peptide Structure1° structure= amino acid sequence2°, 3°, 4° structure= three-dimensional conformation of the protein
3-D structure determinationX-ray crystallography- method of choice. Major limitation
is that the protein must form suitable crystals and the crystal diffraction pattern must be solved
multi-dimensional NMR- technology limited, restricted to peptides and “small proteins” (~ 30 KD, ~ 250 AA’s)
74
Pulsed NMR Techniques:Z
X
Y
EM pulse “tips” themagnetization 90 °into the XY-plane
The magnetization precesses in the XY-plane at the Larmor frequency of the nuclei, which is directly related to the chemical shift (δ) of the nuclei
The magnetization isdetected in the X-axis
The magnetization will relax (recover) back tothe Z-axis. As the magnetization precessesin XY-plane, it “spirals” back to the Z-axis.
relaxation(recovery)
Z
X
Y
Z
X
Y
Animation: http://mutuslab.cs.uwindsor.ca/schurko/nmrcourse/animations/eth_anim/puls_evol.gif
38
75
time
Free Induction Decay (FID)- time domain NMR
In pulse (FT) NMR, all nuclei are tipped at the same time and the FID’s are superimposed.
76
Two-dimensional NMR: a second pulse is after a time delay gives asecond time domain.
Z
X
Y
Z
X
Y
Pulse
precession
relatation
Z
Y
SecondPulse
Z
Y
precess and
relax
first time domain
second time domain
39
77
Correlated NMR spectroscopyCOSY- able to deconvolute all through-bond couplings in a
single experimentNOSY- nuclear Overhauer effect (NOE): provides spacial
(conformational) information from through-space interaction between nuclei.
NOE’s: enhancement of an NMR resonance by polarization transfer through space from a nuclei being irradiated. The effect drops of by 1/r6. Nuclei must typically be within 5 Å.
strong NOE, nuclei within 2.5 Åintermediate NOE, nuclei within 3.5 Åweak NOE, nuclei within 5 Å
Structure calculated according to distance restraints and energy
78
O O
OAcOOAc
AcOOCH3
AcOOAc
OAc
1
1'2'
3'
4' 5'
6'
24
5
3
1H NMR spectrum
13C NMR spectrum
40
79
COSY
1H
1H
O O
OAcOOAc
AcOOCH3
AcOOAc
OAc
1
1'2'
3'
4' 5'
6'
24
5
3
80
1H spectra
13C Spectra
1 or 1' CH2CH2
O O
OAcOOAc
AcOOCH3
AcOOAc
OAc
1
1'2'
3'
4' 5'
6'
24
5
3
HMQC
41
81
X-ray crystallography• x-rays are scattered by electron clouds of atoms in moleculesto give a diffraction pattern. The molecules must be arranged in a ordered crystal.• electron density maps are calculated from the diffraction pattern• electron density map is matched to the amino acid side chain;the primary structure must be known.• limiting step: must obtain suitable crystals of the protein.
Diffraction Pattern follows Bragg’s Law: nλ = 2d sin θdirectx-ray
diffractedx-ray
d
Measure the intensityof the defracted x-rays
Constructive interference(re-enforced diffraction patterns)
82
Proteins have a native three-dimensional conformation (folded state)
Denatured: unfolded state of the protein
Unfolded(denatured)
Native(folded)
ΔG — -16 - 40 KJ/ mol (- 4 - 10 kcal/mol)Keq — 1000 - 108
~~~
~
Proteins folds by stabilizing desired conformations and destablizingundesired ones
ΔS is highly negative
Solvation issuesH-bondinghydrophobic effects
Protein folding is a very complicated problem !!
42
83
The Amide Bond
N H
O
H3C
CH3
N H
O
H3C
CH3
Methyl groups are not equivalent becauseof restricted rotation about the amide bond
N
O
N
O
H
N
R
R
O
H
!
"
#
coplanar
amide bond has a largedipole moment ~ 3.5 Debye
H2O = 1.85 DNH3 = 1.5 DH3CNO2 = 3.5
N
O
N
R
H
H
R
O
N
O
H
N
R
R
O
H
Syn Anti
~80 KJ/mol(19 Kcal/mol)
N
O NHON
O
N H
O
Syn Anti
For Proline the anti rotomer is slightly favored
84
Hydrogen Bonding: non-covalent interaction, 4-16 KJ/mol
O H O H
!- !+
N H N H
!- !+
C O C OC O
!-!+
O
O
H
O
O
H!+
!+
!+
!+
!-
!-
!-
!-
In solution, carboxylic acids exitas hydrogen bonded dimers
NN
O
H
R
O
H
NN
O
H
R
O
H
N-O distance 2.85 - 3.20 Å optimal N-H-O angle is 180 °
43
85
Hydrophobic Effects: tendency for non-polar solutes to aggregate inaqueous solution to minimize the hydrocarbon-waterinterface
Water is a dynamic hydrogen-bonded network. water molecules around a solute is highly ordered- ΔS, entropic penalty (iceberg effect)
Proteins fold to minimize their surface contact with water
micelle structure: hydrocarbon on the inside, polar groupon the outside.
Hydrophobic effects are important in the binding of substrates (ligands)into protein receptors and enzymes
86
Micelles
OP
O
O O
N
dodecylphosphocholine (DPC)
polar headgroup
hydrophobic tail
O
O
Steric acid
44
87
Salts can modify the hydrophobic effect through the change of water structure
H HO
H
HO
H
HO
H
H
O
H
HO
H
H
O
H
H
O
H HO
H
HO
H
H
O
H
H
O
H
H
O
Li+
H
H
O
HHO
HHO
H
H
O
- Cl
HHO
H
H
O
H
HO
LiCl
Dissolving LiCl in water causes a net decrease in overall volume, less“cavities” in bulk water structure for solutes. (salting out)
Other salts such as guandinium chloride break up water structure andcreate more “cavities” or allow “cavities” to form more easily,allowing easier solvation of solutes. (salting in)
Surface tension studies to not support the cavitation theory.
88
Hydrophobic effects are very important in the binding of a substrate into a protein (enzyme or receptor)
Denatured proteins- unfolding of the native three-dimensional structure of a protein by chemical influences such as:
• additives: guandinium salts, urea• heat• pH
old idea: denaturants such as urea unfolded proteins by hydrogen-bonding to the amide backbone
Mechanism probably involves bettersolubilizing the sidechains that arenormally folded into the interiorof the protein
NN
O
H
R
O
H
N NH
H
O
H
NN
O
H
R
O
H
R
H
N NH
H
O
H
H
45
89
Aqueous (hydrophobic) Diels-Alder ReactionDiels-Alder rxn is usually insensitive to solvent effects
HH
O
O
HH
exo
endo
minor
major
O
O
H
H
Endo transition state is more compact
isooctane krel= 1 endo:exo= 4:1methanol 12 8.5:1water 750 25:1
OH
H
H
O
N-Et
O
OH
N-Et
O
O
+
O
H
H
O
N-Et
O
H
isooctane krel= 1.0 methanol 0.5 water 32.04M LiCl 62.02M Guanidinium Cl 16.0
90
Protein Structure:primary (1°) structure: the amino acid sequencesecondary (2°) structure: frequently occurring substructures
or foldstertiary (3°) structure: three-dimensional arrangement of all
atoms in a single polypeptide chainquarternary (4°) structure: overall organization of non-covalently
linked subunits of a functional protein.
Primary structural motifs: α-helixβ-sheetβ-turndisulfide bonds
46
91
α-Helix: amino acids wound into a helical structure3.6 amino acids per coil, 5.4 Å
δ+
δ-
netdipole
N
R
O
H
N
R
O
H
loop
α-helix are connected by loopspdb code: 2A3D
α-helix has a net dipole
CO2-
+H3N
5.4 Å
92
X-ray Structure of Myoglobin
pdb code: 1WLA
47
93
Hydrophobic and Hydrophilic Residues of Myoglobin
Arg
Asp, GluIle
Val
94
Myoglobin
Pro • Ile • Lys • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His • Ser • Lys
48
95Leu Ile Val Phe
pdb code: 1AP9
Bacteriorhodopsin
Schiff base linkage betweenLys-216 and retinal
96
Helical Bundles: hydrophobic sidechains form an interface between α-helices (de novo protein design)
GLY GLU VAL GLU GLU LEU GLU LYS LYS PHE LYS GLU LEU TRP LYS GLY PRO ARG ARG GLY GLU ILE GLU GLU LEU HIS LYS LYS PHE HIS GLU LEU ILE LYS GLY
pdb code: 1qp6
49
97
a
b
c
d
e
f
g
GLY GLU VAL GLU GLU LEU GLU LYS LYS PHE LYS GLU LEU TRP LYS GLY PRO ARG ARG
GLY GLU ILE GLU GLU LEU HIS LYS LYS PHE HIS GLU LEU ILE LYS GLY
a b c d e f g a b c d e f a b c d e f g a b c d e f g a
98
β-sheets and β-turnsparallel anti-parallel
NN
O
R
H
O
NN
R
O
H
R
N
NN
N
H
OR
H
O
H
R
R
H
O
O
R
R
H
H
O
N
H
O
loopor
turnanti-parallelβ-sheet
loopor
turncrossover
50
99
O
NO2C
R(i+3)
HN
O
R(i+2)
O
R(i+1)NH
O
H
N
R(i)
H
O
H3N+
_
H-bond between (i) and (i+1) residues(i)
(i+1)
(i+2)
(i+3)
!-Turn of Lysozyme (residues: Asn46-Thr47-Asp48-Gly49)
(i+1) carbonyl on the opposite side of the sidechains= Type I !-turn
β-Turn: a region of the protein involving four consecutive residues where the polypeptide chain folds back on itself by nearly 180 °. This chain reversal gives proteins a globular rather than linear structure. (Chou & Fasman J. Mol. Biol. 1977, 115, 135-175.)
β-Turn
100
!-Turn of Lysozyme
pdb code: 1AZF
Typ53-Asp52-Thr51-Ser50-Gly49-Asp48
Thr43-Asn44-Arg45---------Asn46-Thr47
51
101
Anti-parallel β-sheets of lectinpdb code: 2LAL
Parallel β-sheets carbonic anhydrase
pdb code: 1QRM
102
Disulfide bonds: covalent structural scaffolds, redox active, reversible
2 Cys-SH Cys-S-S-Cys (Cysteine)[O]
[H]
Chain B: 30 AA’s
Cys(B)19-Cys(A)20
Cys(A)6-Cys(A)11
Cys(B)7-Cys(A)7
Chain A: 21 AA’s
Human InsulinEGF
pdb code: 1XDApdb code: 3EGF
Cys14-Cys31Cys6-Cys20
Cys33-Cys42
Somatostatin Analogpdb code: 1SOC
52
103
lysozymepdb code: 1AZF
Cys6 - Cys127
Cys30 - Cys115
ribonucleasepdb code: 1ALF
Cys58 - Cys110
Cys65 - Cys72
Cys26 - Cys84
Cys40 - Cys95
Disulfide bonds
Cys76 - Cys94
Cys64 - Cys80
His 119
His12
104
Somatostatin:S
S
Cys-Ser-Thr-Phe-Thr-Lys
Ala-Gly-Cys-Lys-Asn-Phe-PheTrp
!-turn reponsible for
biological activity
N
O NH
Ph
O
OH
O
NH
O
HN
O
Ph
NH
O
NH2
NH
Pro-Phe-D-Trp
Phe-Thr-Lys
D-stereochemistryat the (i+2) psoitionstabilizes the !-turn
remove disulfide
N
O NH
Ph
O
OH
O
NH
O
HN
O
Ph
NH
O
NH2
NH
H3C
same activity as somatostatin
N
O NH
Ph
O
O
NH
O
HN
ONH
O
NH2
NH
H3C
OH
100x more potentthan somatostatin
Recommended