Protein structure (and function)

Protein structure (and function)

1. Primary structure

   • How do we find primary structure?
     • mostly inferred from DNA sequence
       • Prediction is based on the need for amino acids to be in a certain position in order to achieve the desired structure
         • Protein databases can provide a wealth of predictions
           • Sequence alignment
           • Domain composition
           • post-translational modifications
           • protein-protein interactions
           • structure
           • etc.
     • Can be obtained directly by amino acid sequencing using edman degradation
     • Or using mass spectrometry

2. Secondary structure

   • Alpha-helix
     • Space-filling
     • Backbone
     • Sticks
     • Ribbon
   • Experimental determination of Protein Secondary Structure by Circular Dichroism (CD)
     • CD spectroscopy in the "far-uv" spectral region (190-250 nm) reveals secondary structure.
     • Alpha-helix, beta-sheet, and random coil each give a characteristic shape of CD spectrum.
     • The fraction of each secondary structure type in any protein can be calculated from its far-uv CD spectrum.
     • This gives a % for each secondary structure element but no information on arrangement.

3. Tertiary structure

   • The way in which individual secondary structural elements; α-helices, β-sheets and random coil, pack together within a protein and between sub-domains of a protein
   • Information on tertiary Structure from CD
     • The CD spectrum of a protein in the "near-uv" spectral region (250-350 nm) gives some information on tertiary structure
     • CD signals of aromatic amino acids and disulfide bonds, are sensitive to the overall tertiary structure of the protein.
     • Their absorbance is affected by the local ‘environment’ and can be observed dynamically.
     • Protein unfolding or ‘melting’ can be followed by CD at different temperatures.

4. Quaternary structure

   • Quaternary structure is the relationship between individual proteins in a multimeric complex

5. Folding of a peptide into a protein

6. Proteins are held together by different ionic interactions

   • Ionic interactions
     • Attraction between +ve and -ve charged ions
   • Van der waals
     • Short range weak electrical attraction and repulsion
   • Hydrogen bonds
     • Involve a H shared between O and N atoms

7. Other techniques

   • X-ray crystallography
   • NMR nuclear magnetic resonance
     • In most atoms the ‘spin’ of subatomic particles are paired against each other, such that the nucleus of the atom has no overall spin.
     • However, in some atoms (such as 1H,13C,15N) there are uneven numbers of protons and neutrons so the nucleus has a slight wobble in the spin.
       • Proteins are produced recombinantly, usually in bacteria grown in media where the sole nutrient source is 15N and/or 13C, so that all protein produced are singly or doubly labelled with 15N and/or 13C in every atom
     • ‘NMR active’ nuclei (like 1H or 13C) resonate at a specific frequency in a strong magnetic field.
     • Depending on local environment, different protons resonate at slightly different frequencies, known as a chemical shift.
     • NMR structure determination is an iterative process, so one arrives at several possible structures which are usually represented as an ensemble.
   • Electron microscopy
     • EM uses negative stain (shown) or vitreous ice (cryo-EM) to preserve the specimen.
       • Image analysis is then employed to build up an average structure.
         • The more ordered and more symmetrical the structure, the easier the averaging process. e.g. actin has helical symmetry, viruses have radial symmetry

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