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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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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


Edman degradation of oligopeptidesTheory of automatic sequencing


Liquid chromatogram of the PTH derivatives of 20 amino acids

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


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% R1 100% R1step 2: 95% R2 + 5% R1 99% R2 + 1% R1step 10: 63% R10 + 30% R9 91% R10 + 8% R9step 20: 38% R20 + 38% R19 + 18% R18 83% R20 + 16% R19step 50: 8% R50 + 21% R49 + 26% R48 61% R50 + 30% R49 + 7% R48



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


known

some uncertainty

some uncertainty

known

some uncertainty

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


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


MALDI-TOF MS:

Matrix AssistedLaser DesorptionIonization -

Time Of FlightMass Spectrometer


ESI MS: ElectroSpray Ionization Mass Spectrometer


MS-MS or Tandem Mass Spectrometry


  • 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


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


  • 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


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 


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






X-ray diffraction structures

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


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


Preparation of single crystals structures

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



1 structuresH NMR spectrum of lysozyme


1 mM [ structures13C-15N] Ubiquitin in 90% H2O-10% D2O13C-1H HSQC - An example for interaction through a chemical bond


1 mM [ structures13C-15N] Ubiquitin in 90% H2O-10% D2OHNCA - An example for interaction through multiple bonds



X ray vs nmr

Diffraction space: 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


  • Stability: space

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

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

  • disulfide bridges mercaptoethanol, DTT

  • H-bonds pH extremes

  • hydrophobic interactions detergents, urea

  • ionic interactions changing pH or ionic strength


Peptides proteins
Peptides space Proteins


Motif: spaceA distinct folding pattern forelements of secondary structure;

also called a fold orsupersecondary structure.


Domain space

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


Subunit space

Separate polypeptide chains of the same protein


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


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

Reasons and mechanismare not understood


Proteins
Proteins structures

  • Functions:

  • catalysis enzymes

  • transport and storage myoglobin, hemoglobin

  • motion actin myosin

  • defense skin and hair proteins

  • regulation hormones, exp. factors

  • fuel e.g. in plant seeds


Proteins1
Proteins structures

  • Functions:

  • CATALYSIS ENZYMES

  • transport and storage myoglobin, hemoglobin

  • motion actin myosin

  • defense skin and hair proteins

  • regulation hormones, exp. factors

  • fuel e.g. in plant seeds


Enzyme Classification structures

1 Oxidoreductases (electron, hydride ion, H atom)

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

3 Hydrolases (functional groups  water)

4 Lyases (formation or saturation of double bonds)

5 Isomerases

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


Each enzyme name ends - structuresase, 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


Some enzymes with inorganic ions as cofactors structures

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

Iron-sulfur proteins succinate dehydrogenase, aconitase, dinitrogenase

Copper(II) cytochrome oxidase, superoxide dismutase

Zinc(II) alcohol dehydrogenase, superoxide dismutase

Magnesium hexokinase, glc-6-phosphatase, pyruvate kinase

Potassium pyruvate kinase

Molibdenum dinitrogenase

Selenium glutathione peroxidase


Electron, atom or group transfer coenzymes structures

NAD+ hydride ion nicotinic acid (niacin)

FAD+ electron riboflavin (B2)

CoA ac(et)yl group pantothenic acid

TPP aldehyde group thiamine (B1)

pyridoxal phosphate amino group pyridoxine (B6)

coenzyme B12 H atoms, alkyl groups (B12)

ubiquinone electron

tetrahydrofolate one-carbon groups folic acid


Proteins2
Proteins structures

  • Functions:

  • catalysis enzymes

  • TRANSPORT AND STORAGE MYOGLOBIN, HEMOGLOBIN

  • motion actin myosin

  • defense skin and hair proteins

  • regulation hormones, exp. factors

  • fuel e.g. in plant seeds



Proteins3
Proteins structures

  • Functions:

  • catalysis enzymes

  • transport and storage myoglobin, hemoglobin

  • MOTION ACTIN, MYOSIN

  • defense skin and hair proteins

  • regulation hormones, exp. factors

  • fuel e.g. in plant seeds


Proteins4
Proteins structures

  • Functions:

  • catalysis enzymes

  • transport and storage myoglobin, hemoglobin

  • motion actin myosin

  • DEFENSE SKIN AND HAIR PROTEINS

  • regulation hormones, exp. factors

  • fuel e.g. in plant seeds



Collagen structures


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


Silk fibroin residues,

Spider’s net


Proteins5
Proteins residues,

  • Functions:

  • catalysis enzymes

  • transport and storage myoglobin, hemoglobin

  • motion actin myosin

  • defense skin and hair proteins

  • regulation hormones, exp. factors

  • fuel e.g. in plant seeds


Online resources: residues,

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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