Frederick sanger 1918
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1958 Nobel Prize in Chemistry: for his work on the structure of proteins, especially that of insulin. GIVEQCCASVCSLYQLENYCN PVNQHLCGSHLVEALYLVCGERGFFYTPKA. Frederick SANGER (1918-).

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Frederick SANGER (1918-)

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Frederick sanger 1918

1958 Nobel Prize in Chemistry:for his work on the structure of proteins,especially that of insulin

GIVEQCCASVCSLYQLENYCNPVNQHLCGSHLVEALYLVCGERGFFYTPKA

Frederick SANGER(1918-)

1980 Nobel Prize in Chemistry (1/4):for his contribution concerning thedetermination of base sequencesin nucleic acids


Protein sequencing

Protein sequencing

  • Edman degradationup to ~20-30 residues, time consuming,needs large amount of purified protein

  • Recombinant DNA technologyfast, but it does not consider splicing andposttranslational modifications

  • Mass Spectrometrynot for exact sequencing of long chains, rather for cataloging of cellular proteins


Frederick sanger 1918

Edman degradation of oligopeptidesTheory of automatic sequencing


Frederick sanger 1918

Liquid chromatogram of the PTH derivatives of 20 amino acids

Derivatives ofdifferent aminoacids can be distinguished AND identified bytheir elution times.


Frederick sanger 1918

Possible problems with Edman degradation:

- multiple peptide chains:the primary result of sequencing of the native protein consisting ofn peptide chains would be n amino acids for each positionin the sequence  it is impossible to decide which amino acid follows which

- imperfection (<100% efficiency):

if cleavage is performed with 95% OR 99% efficiency, the result ofstep 1:100% R1100% R1step 2:95% R2 + 5% R199% R2 + 1% R1step 10:63% R10 + 30% R991% R10 + 8% R9step 20:38% R20 + 38% R19 + 18% R1883% R20 + 16% R19step 50:8% R50 + 21% R49 + 26% R4861% R50 + 30% R49 + 7% R48


Frederick sanger 1918

Irreversible cleavage of cystine bridges


Frederick sanger 1918

  • Recombinant DNA technologythe human genome is “known”and the DNAamino acid dictionary is known, too

However,the encoded, the nascent and the nativesequences are NOT necessarily the same


Frederick sanger 1918

known

some uncertainty

some uncertainty

known

some uncertainty

…and this is what we wouldlike to figure out...


Frederick sanger 1918

Nobel Prize inChemistry2002(1/4 - 1/4)

John B. FENN

Koichi TANAKA

for their development of soft desorption ionisation methods formass spectrometric analyses of biological macromolecules


Frederick sanger 1918

MALDI-TOF MS:

Matrix AssistedLaser DesorptionIonization -

Time Of FlightMass Spectrometer


Frederick sanger 1918

ESI MS: ElectroSpray Ionization Mass Spectrometer


Frederick sanger 1918

MS-MS or Tandem Mass Spectrometry


Frederick sanger 1918

  • What can amino acid sequence be used for?

  • Searching for similarities by comparison to known sequences classification among protein types (FUNCTION)

  • Searching for similarities by comparison to other species evolutionary consequences can be drawn

  • Searching for internal repeats history of an individual protein

  • Searching for signals designating destination or process control  fate of the protein between translation and native state

  • Sequence data basis for preparing antibodies specific to the protein

  • Sequence data  reverse genetics:making DNA probes for the genes encoding the proteins

  • Sequence data  structures of higher order


Frederick sanger 1918

Robert Bruce MERRIFIELD1984, Nobel Prize in Chemistryfor his development ofmethodology forchemical synthesison a solid matrix

1955, VIGNEAUD:

Oxytocin: the first syntheticpeptide hormone

(“conquering the Himalayas”)

1902, Emil FISCHER:The first artificial peptide bond


Frederick sanger 1918

  • What can you use synthetic peptides for?

  • Antigens to stimulate the formation of specific antibodies

  • Isolation of receptors for hormones and signalling molecules (affinity cromatography)

  • Drugs(e.g. hormone analogs)

  • Study of these can help define the rules governing the 3D structure of proteins


Frederick sanger 1918

Peptide bonds are rigid:torsion does not occur around C(O)-NHbonds

Extent of torsion around N-C bond is denoted by dihedral angle whereas that around C-C(O) bond is denoted by dihedral angle 


Frederick sanger 1918

Due to sterichindrance=0 AND =0cannot occur.Only certain pairsof values are permitted by thegeometries of thesuccessive peptidebonds


Frederick sanger 1918

Ramachandran - plot for Ala


Frederick sanger 1918

Typical occurrences of each amino acids in secondary structures


Frederick sanger 1918

Ramachandran plot of pyruvate kinase (except Gly’s)


Frederick sanger 1918

Human Serum Albumin would look like this, if...


Frederick sanger 1918

X-ray diffraction

X-rays are scattered byelectrons around nucleiScattering pattern can beused for calculation of the positions of nucleiThe sample is asingle crystal


Frederick sanger 1918

Signals of interactions over the space help us to turnthe sequence into 3D structure


Frederick sanger 1918

Preparation of single crystals

In most cases it is by far not easy as it seems...


Frederick sanger 1918

Nuclear Magnetic Resonance (NMR)


Frederick sanger 1918

1H NMR spectrum of lysozyme


Frederick sanger 1918

1 mM [13C-15N] Ubiquitin in 90% H2O-10% D2O13C-1H HSQC - An example for interaction through a chemical bond


Frederick sanger 1918

1 mM [13C-15N] Ubiquitin in 90% H2O-10% D2OHNCA - An example for interaction through multiple bonds


Frederick sanger 1918

Nuclear Overhauser Effect (NOE) - Interaction through the space


X ray vs nmr

Diffraction: interaction with electron density

Sample: single crystal

Result: a “sharp” static snapshot with good spatial resolution

Perutz and Kendrew (1962)

Resonance: interaction with magnetic moments

Sample: isotope labelled protein

Result: a “blurry”dynamic picture of a conformational ensemble”

Wüthrich (2002) (1/2)

X-ray vs NMR


Frederick sanger 1918

  • Stability:

  • “enthalpy side”: formation of bonds“entropy side”: rearrangement of solvent “structure”

  • Chemical bonds participating in stabilizing protein structureand agents used to cleave them:

  • disulfide bridgesmercaptoethanol, DTT

  • H-bondspH extremes

  • hydrophobic interactionsdetergents, urea

  • ionic interactionschanging pH or ionic strength


Peptides proteins

Peptides  Proteins


Frederick sanger 1918

Motif:A distinct folding pattern forelements of secondary structure;

also called a fold orsupersecondary structure.


Frederick sanger 1918

Domain

A distinct structural unit of a polypeptide;they may have sparate functions andthey may fold as independent, compact units


Frederick sanger 1918

Subunit

Separate polypeptide chains of the same protein


Frederick sanger 1918

The same short sequence may take different secondary structuresdepending on its broader environment


Frederick sanger 1918

Prion:(proteinaceous infectious only)upon dimerization it suffersdramatic conformational changeleading to spongiformdegeneration (CJD)

Reasons and mechanismare not understood


Proteins

Proteins

  • Functions:

  • catalysisenzymes

  • transport and storagemyoglobin, hemoglobin

  • motionactin myosin

  • defenseskin and hair proteins

  • regulationhormones, exp. factors

  • fuele.g. in plant seeds


Proteins1

Proteins

  • Functions:

  • CATALYSISENZYMES

  • transport and storagemyoglobin, hemoglobin

  • motionactin myosin

  • defenseskin and hair proteins

  • regulationhormones, exp. factors

  • fuele.g. in plant seeds


Frederick sanger 1918

Enzyme Classification

1Oxidoreductases(electron, hydride ion, H atom)

2Transferases(group transfer, e. g. phosphate, -COO, methyl)

3Hydrolases(functional groups  water)

4Lyases(formation or saturation of double bonds)

5Isomerases

6Ligases(C-C, C-S, C-O, C-N bonds, for ATP)


Frederick sanger 1918

Each enzyme name ends -ase, except...

Each enzyme is (basically) protein, except…

The enzyme exerts its activity in the native conformation,and the reaction takes place at the active site

Enzymes do not usually contain protein only:

apoenzyme (protein) + X = holoenzyme

X = cofactor

cofactor = inorganic ion or coenzyme (or both)

The cofactor bound to the peptide chain by covalent bond is called

prosthetic group


Frederick sanger 1918

Some enzymes with inorganic ions as cofactors

Iron(II)-iron(III)cytochrome oxidase, catalase, peroxidase

Iron-sulfur proteinssuccinate dehydrogenase, aconitase, dinitrogenase

Copper(II)cytochrome oxidase, superoxide dismutase

Zinc(II)alcohol dehydrogenase, superoxide dismutase

Magnesiumhexokinase, glc-6-phosphatase, pyruvate kinase

Potassiumpyruvate kinase

Molibdenumdinitrogenase

Seleniumglutathione peroxidase


Frederick sanger 1918

Electron, atom or group transfer coenzymes

NAD+hydride ionnicotinic acid (niacin)

FAD+electronriboflavin (B2)

CoAac(et)yl grouppantothenic acid

TPPaldehyde groupthiamine (B1)

pyridoxal phosphateamino grouppyridoxine (B6)

coenzyme B12H atoms, alkyl groups(B12)

ubiquinoneelectron

tetrahydrofolateone-carbon groupsfolic acid


Proteins2

Proteins

  • Functions:

  • catalysisenzymes

  • TRANSPORT AND STORAGEMYOGLOBIN, HEMOGLOBIN

  • motionactin myosin

  • defenseskin and hair proteins

  • regulationhormones, exp. factors

  • fuele.g. in plant seeds


Frederick sanger 1918

See also the Molecular Tutorial


Proteins3

Proteins

  • Functions:

  • catalysisenzymes

  • transport and storagemyoglobin, hemoglobin

  • MOTIONACTIN, MYOSIN

  • defenseskin and hair proteins

  • regulationhormones, exp. factors

  • fuele.g. in plant seeds


Proteins4

Proteins

  • Functions:

  • catalysisenzymes

  • transport and storagemyoglobin, hemoglobin

  • motionactin myosin

  • DEFENSESKIN AND HAIR PROTEINS

  • regulationhormones, exp. factors

  • fuele.g. in plant seeds


Frederick sanger 1918

What do hairdressers do when they make a permanent wave?


Frederick sanger 1918

Collagen


Frederick sanger 1918

Collagen helices are cross-linked, mostly by hydroxyproline residues,to the formation of which ascorbic acid (vitamin C) is indispensable  that is why the lack of vitamin C caused scurvy


Frederick sanger 1918

Silk fibroin

Spider’s net


Proteins5

Proteins

  • Functions:

  • catalysisenzymes

  • transport and storagemyoglobin, hemoglobin

  • motionactin myosin

  • defenseskin and hair proteins

  • regulationhormones, exp. factors

  • fuele.g. in plant seeds


Frederick sanger 1918

Online resources:

http://bcs.whfreeman.com/lehninger (Ch3, Ch4, Ch5)

see esp. Molecular Tutorial “Protein Architecture”

http://bcs.whfreeman.com/biochem5 (Ch3, Ch4)

For online quizzing give:

[email protected]

as instructor’s e-mail address


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