Peptide bond formation - fwd reaction
Condensation or dehydration reaction
Peptide bond is a substituted amide (-CONH2) linkage
Occurs during translation
High energy cost “directly”
- Keq favours reactant (+delta G)
- costs >4 ATP equivalent to produce each peptide bond in a protein ie. 4 x 30.5 = >122 kJ/mole
Considerable “indirect” costs in terms of cellular resources
Peptide bond hydrolysis - rev reaction
Exergonic - delta G is neg, so spontaneous
Free energy is only -21kJ/ mole
High activation energy, so occurs slowly
Peptide bonds therefore chemically stable
Protein turnover
Means of controlling an enzymes activity (biological function) in a cell: protein synthesis and deliberate degradation offers a rapid mechanism for metabolic control, admittedly at the expense of destroying proteins that cost s lot of energy to synthesise in the first place
- Transcription (DNA >mRNA) eg transcription factors
- Translation (mRNA> protein) eg life span of mRNA
- Activation/ inactivation of pre-existing enzyme eg kinases
- Degradation eg targeted hydrolysis
- Enzyme kinetics eg michaelis - menten response to changing (S)
Charges on polypeptides and proteins
The overall charge on a polypeptide or protein at a particular pH is the sum of the charges on all of its ionisable groups at that pH
Consider:
- pKa of single alpha amino group
- pKa of the single alpha carboxylic group
- pKas of any ionisable R groups present
Isoelectric point (IEP or pI) of a polypeptide of protein
The pH at which its net charge is zero
Can be calculated as the weighted average of the pKas of all of the ionisable groups present.
Amino acid composition of proteins
The relative proportions of amino acids in proteins are highly variable:
- amino acid composition determined by acid- hydrolysing polypeptide to cleave peptide bonds prior to analysis, but:
- destroys tryptophan (Trp)
- converts the amides asparagine to aspartate so alternative methods needed to measure these
Each protein has characteristic composition of AA
In a particular protein, some aa mah be under represented of absent
In general, small AAs predominate
- proteins exhibit broad range of IEPs
Protein structure general considerations
Conformation- special arrangement of all the atoms in a protein
- by rotation about single covalent bonds
- without breaking covalent bonds
- protein can assume unlimited conformations
However, each protein had a specific biological function which strongly suggest a unique structural form or shape and can also be crystallised, also suggesting unique structures.
Therefore, proteins tend to be in their thermodynamically most stable arrangement, ie those with the lowest free energy G
Proteins in this folded and functional form are called native proteins and are said to be in their native conformation.
Protein structure underlying themes
- pure protein had a unique 3D structure
- structure is determined solely by the AA sequence of the protein
- biological function of a protein depends on its structure
- common structural patters emerge and recognising them can simplify our understanding of protein structure and architecture
- structure help together by strong covalent linkages, but stabilised by weak non-covalent interactions such as H bonds, hydrophobic interactions, ionic or electrostatic interactions, dipole dipole interactions, Van der Waals forces
Hierarchy of protein structure
- primary structure
Linear sequence of amino acids attached through covalent peptide bonds or disulphides bonds, starting from N terminus (left) and ending at C terminus (right) - secondary structure
Formation of stable 3D structural entities that have recognisable shapes and features in sections of the polypeptide chain. Involve relatively short-range interactions that develop within relatively small and localised regions of the primary sequence. Contain a co-planar conformation, and steric hindrance- this rigid feature greatly reduced the range of conformations the protein can assume. Form alpha helix (spring form), beta (sheets) - tertiary structure
Overall arrangement of all atoms in a protein molecule. Describes the way in which multiple elements of secondary structure come together in 3D space following folding. Hydrophobic interactions are the major force driving protein folding and stabilising the tertiary structure. - Quaternary structure
Two or more polypeptide chains that associate non-covalently to form a fully functional protein complex
Fibroids proteins
Polypeptides arranged in long rope- like strands or sheets. Usually a single type of secondary structure. Provide support, shape, external protection
Eg. Keratin of hair, feathers, nails
Globular proteins
Polypeptides folded into a more compact structure, often roughly spherical in shape. Often contain several types of secondary structure. Chains typically fold back on themselves leading to broad structural diversity. Structural diversity provides functional diversity
relationship between number of AA and molecular weight
Difficult to determine actual molecular weight of a protein, but can approximate:
- approx size of AA residue is 110Daltons
- so to calculate size:
MW of protein= no. Of residues x 110Da
Secondary structure features
- co planar
- O of carbonyl and H of amide are trans to eachother
- c=n bond is locked so no free movement, but the atoms on either side can still move freely
- steric hindrance shows flexibility of bonds
Alpha helix secondary structure
- simplest arrangement
- rigid peptide bonds but rotation around other bonds
- polypeptide backbone wound around imaginary longitudinal axis through centre
- R groups point outwards
- approx 4 amino acids per turn
- each residue is therefor rotated 100degrees from the previous
- right handed turns
- H bonds stabilise structure
Destabilising-
- long stretches of the same ionisable AAs
- AAs with bulky side chains
- presence of glycine and proline
Beta sheets and strands secondary structure
- single strand alone not stable, multiple strands arranged side by side with H bonds between the sheets
- high gly, pro and ala contents
- R groups have little involvement in stabilisation
- beta turns which reverse direction for compact globular proteins
- join ends of anti parallel sheets, forms 180 turn, involves 4 amino acids
