24

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