Bioinformatics

Biological Basics

1. Amino acids I 2. Peptide bond I 3. Proteins I 4. DNA I 5. Miscellaneous

3. Proteins

• macromolecules / polymers
• 'workhorses of the cell'
• made up of various L-amino acids connected by peptide bonds
• linear chains (unbranched)
  • protein molecule that consists of a single polypeptide chain is said to be monomeric
  • proteins made up of more than one polypeptide chain are called oligomeric
• interact with other proteins, nucleic acids, small molecules, ions, etc.

Protein Denaturation

• organized molecular configuration is disturbed

Main causes of denaturation
changes in temperature	heat increases the kinetic energy (energy of motion) of the protein chain excessive motion can break relatively weak H-bonds, electrostatic interactions and hydrophobic interactions protein chain is free to rearrange after disrupting
changes in pH	change electric charge of acidic or basic functional groups on the protein disrupt or create electrostatic interactions that will alter the protein structure pH 2 carboxylic acid groups are not charged pH 7 carboxylic acid groups are negatively charged (-COO-) and amino groups are positively charged (-NH3+) pH 12 amino groups are not charged
changes in salt concentration	high salt concentrations reduce electrostatic interactions extra ions in solution tend to "insulate" charges on the protein low salt concentrations increase electrostatic interactions Hofmeister series describes the relative effects of some anions and cations in precipitating proteins cations / anions effect = independent and additive (anion has a larger effect !) anions / cations on the left are most effective in precipitating proteins ("salting-out") those on the right are most effective in solubilizing proteins ("salting-in") chaotropic anions = water-structure breakers, protein destabilizer (large ions, high polarizability) kosmotropic anions = polar water-structure makers, protein stabilizer (usually small ions, low polarizability) ammonium sulfate (NH4)2SO4 is the most frequently used salt for salting-out experiments above 75% of protein impurities can be removed by precipitation (see also chapter: protein purification - salt precipitation) dividing line: Na+ and Cl- [neutral with respect to stabilizing/destabilizing proteins] cations NH4+ > K+ > Na+ > Li+ > Mg2+ > Ca2+ > guanidium > urea anions SO42- > HPO42- > OH- > F- > CH3COO- > citrate > tartrate > Cl-> Br- > NO3- > ClO3- > I- > ClO4- > SCN- The Hofmeister Series (by M. Chaplin, London South Bank University) Kosmotropes and Chaotropes (by M. Chaplin, London South Bank University) Chi, E.Y.; Krishnan, S.; Randolph, T.W.; Carpenter, J.F.; Pharm. Res. 2003, 20(9), 1325-1336. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation. [PubMed]
detergents	detergents are amphiphilic molecules (both hydrophobic and hydrophilic parts) disrupt hydrophobic interactions hydrophobic parts of the detergent associate with the hydrophobic parts of the protein (coating with detergent molecules) hydrophilic ends of the detergent molecules interact favorably with water (nonpolar parts of the protein become coated with polar groups that allow their association with water) hydrophobic parts of the protein no longer need to associate with each other dissociation of the non-polar R groups can lead to unfolding of the protein chain (same effect as in a nonpolar solvents)
H-bonding agents (urea, guanidine ...)	H-bonding is important in maintaining secondary, tertiary and quaternary structure H-bonding agents compete with H-bonding between protein functional groups R groups that normally associate due to H-bonding, no longer need to associate dissociation can lead to unfolding of the protein chain urea guanidinium chloride
oxidants & reductants	mild reductants and mild oxidants can lead to changes in protein conformation, that may alter the function of the protein mild reductants can break disulfide bonds may lead to dissociation of parts of the protein chain(s) that are normally associated protein reduction with an excess of ß-mercaptoethanol mild oxidants can cause the formation of disulfide bonds may lead to association of parts of the protein chain that are normally not associated stronger oxidizing and reducing agents can change the nature of protein R groups most easily oxidized R groups next to sulhydryl groups are: phenol (tyrosine), hydroxyl (serine & threonine), amine (lysine, arginine, histidine), sulfide (methionine)
non-polar solvents	disrupt hydrophobic interactions (association of nonpolar R groups) non-polar R groups no longer need to associate, because they can now interact with the solvent dissociation of the non-polar R groups can lead to unfolding of the protein chain
... and also UV / ionizing radiations and mechanical energy

Additional information

• LIFE - The Science of Biology (7th edition) (by W.K. Purves, D. Sadava, G.H. Orians, H. Craig Heller)
• Bennion, B.J.; Daggett, V.; Proc. Natl. Acad. Sci. USA 2003, 100(9), 5142-5147. The molecular basis for the chemical denaturation of proteins by urea. [PDF I PubMed]
• Cozzone, A.J.; Encyclopedia of Life Sciences 2002, 1-10. Proteins: fundamental chemical properties. [PDF]

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