Basic properties of proteins Maria Berggård Silow Methods in Molecular Biology course 2008

1. Basic properties of proteins Maria Berggård Silow Methods in Molecular Biology course 2008

2. Basic properties of proteins I • Amino acids • The peptide bond • Proteins are polymers • Protein Structural organization • Protein stability • Protein folding and dynamics • Natively unfolded proteins • Co-factors and prosthetic groups

3. Basic structure of amino acids • Chiral • L-amino acids • Zwitterionic

4. 20 standard amino acids Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Alanine Arginine Asparagine Aspartate Cysteine Glutamate Glutamine Glycine Histidine Isoleucine

5. Non-standard amino acids Modified after synthesis D-alanine – peptidoglycan, bacterial cell wall Hydroxyproline - collagen Hydroxyserine - collagen GABA - neurotransmitter Dopamine - neurotransmitter Formyl methionine – initiation of translation

6. Non-polar: Alanine Glycine Isoleucine Leucine Methionine Phenylalanine Proline Tryptophan Valine Polar non-charged: Asparagine Cysteine Glutamine Serine Threonine Tyrosine Polar charged: Arginine Aspartate Glutamate Histidine Lysine

7. The peptide bond Partial double bond Planar structure f, y -dihedral angles Determined by the nature of the amino acid side chain

8. Proteins are Polymers • Solvent quality • /temperature • Preferred bond angles • /rotation angles • Excluded volume

9. Protein structural organization • Primary structure • Amino acid sequence • Secondary structures • Dictated by planar structure • of the peptide bond • All H-bonds need to be satisfied • 310 helix • P-helix • W-loop

10. Protein structural organization • Super-secondary structures / Domains • Helix bundle • TIM-barrel • bab-domain

11. Protein structural organization • Tertiary • structure • Quaternary • structure

12. Protein stability Protein stability: DGN-U=DHN-U-TDSN-U

13. Protein stability Stabilizing forces Destabilizing forces Hydrogen bonding Chain entropy Van der Waals interactions Disulfide bonds Hydrophobic effect Typical protein stability: 5-15 kcal/mol Denaturants: temperature pH chemicals (urea, guanidine HCl)

14. Protein denaturation The simplest case: only D and N are populated Keq = [D] / [N] The equilibrium constant in ”water” can be extrapolated log KH20 = log Keq * m[denaturant] Protein stability in ”water” is the free energy of unfolding: DGH2O = -2.3 * RT * logKH2O

15. Protein folding and dynamics • In vitro: • Secondary and tertiary structure forms • simultaneously • Protein topology • Domains dock • In vivo: • chaperones and chaperonins • Dynamics • Proteins are molecular • machines

16. Natively unfolded proteins • >100 proteins natively unfolded • Low hydrophobicity, high net charge • Large hydrodynamic radius • Low secondary structure content • High flexibility

17. Reactive: Asparagine Arginine Cysteine Glutamic acid Glutamine Histidine Lysine Methionine http://www.ebi.ac.uk/thornton-srv/databases/CSA/

18. Structurally important: Proline – helix breaker Glycine - helix breaker Alanine – helix former Isoleucine – helix former Leucine – helix former Valine – helix former

19. Interaction with other molecules: Aspartate Glutamate Histidine Lysine Arginine

20. Aromatic residues Phenylalanine Tyrosine Tryptophan

21. Co-factors and prosthetic groups Ions: Ca2+, Mg2+, Cl- Metal clusters: iron-sulfur Porphyrins: heme, chlorophyll Carotenoids: retinol, violaxanthin NAD+/NADH, NADP+/NADPH ATP, GTP Acetyl-coenzymeA Flavin Biotin Cobalamin Lipoic acid Tetra hydrofolate Thiamine

22. Basic properties of proteins II • Molecular weight • Composition • Aggregation state • Charge • Color

23. Cromatography • HPLC- High Pressure Liquid Chromatography • Reversed phase chromatography • Organic solvents – used for separation • of peptides • FPLC- Fast Protein Liquid Chromatography • Size exclusion • Ion exchange • Hydrophobic interaction • Affinity • Hydroxylapatite

24. Molecular weight • Size exclusion chromatography • Separation according to • hydrodynamic radius • Preparative • Analytical • Electrophoresis • Separation according to molecular weight in • SDS-PAGE • Separation according to molecular weight and • charge in native gel electrophoresis • Analytical • Preparative

25. Molecular weight • Mass spectrometry • MALDI-TOF • ESI • Analytical • Analytical ultra centrifugation • Analytical

26. Composition • Amino acid sequencing • Edman degradation • Tandem mass spectrometry (MS/MS) • Metal analysis • Atomic absorption spectroscopy (AAS) • ICP-MS • Post translational modification • Detection by Bioinformatics / Measurement of MW • HPLC based analysis - glycosylation • Mass spectrometric analysis

27. Aggregation state • Size exclusion chromatography

28. Aggregation state • Light scattering techniques- • Classical light scattering • Dynamic light scattering • Small angle x-ray scattering

29. Aggregation state • Rate zonal centrifugation – • Layer sample on top of premade gradients • Sucrose gradients (size, shape and density) • Preparative • Isopycnic centrifugation – • Mix sample and gradient forming material – Gradients will • form in the centrifuge • CsCl gradients (isopycnic density) • Preparative

30. Charge • Protein charge • Isoelectric focusing • Ion-exchange chromatography • pKa of individual amino acids • NMR

31. Color