24 47 Peptide and Protein Analysis Primary (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 are separated by hplc, then detected by post-column derivatization 1972 Nobel Prize in Chemistry William Stein Stanford Moore peptide -or- protein [H] reduce any disulfide bonds Enzymatic digestion R CO 2 NH 3 individual amino acids -or- H 3 O + , Δ liquid chromatography derivatize w/ ninhydrin Detected w/ UV-vis Different amino acids have different chromatographic mobilities (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 CO 2 NH 3 O O O +
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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)
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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
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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.
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Fluorescent tags Dansyl- detected by UV or fluorescence
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
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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
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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)
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
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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
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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
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
+
+
++
+
- +
++
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
- +
+
+
+
+
+
++
+++++
+++
++++
+++++
++++
+++
++++
+++++
+
+
- +
+
+
+
+
+
+
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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)
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[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
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
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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)
In pulse (FT) NMR, all nuclei are tipped at the same time and the FID’s are superimposed.
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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
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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
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O O
OAcOOAc
AcOOCH3
AcOOAc
OAc
1
1'2'
3'
4' 5'
6'
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5
3
1H NMR spectrum
13C NMR spectrum
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COSY
1H
1H
O O
OAcOOAc
AcOOCH3
AcOOAc
OAc
1
1'2'
3'
4' 5'
6'
24
5
3
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1H spectra
13C Spectra
1 or 1' CH2CH2
O O
OAcOOAc
AcOOCH3
AcOOAc
OAc
1
1'2'
3'
4' 5'
6'
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5
3
HMQC
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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
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 °
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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
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Micelles
OP
O
O O
N
dodecylphosphocholine (DPC)
polar headgroup
hydrophobic tail
O
O
Steric acid
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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.
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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
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Aqueous (hydrophobic) Diels-Alder ReactionDiels-Alder rxn is usually insensitive to solvent effects
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
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X-ray Structure of Myoglobin
pdb code: 1WLA
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Hydrophobic and Hydrophilic Residues of Myoglobin
Arg
Asp, GluIle
Val
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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
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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
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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
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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
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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.)