Protein structure (and function)

Primary structure
1. How do we find primary structure?
  1. mostly inferred from DNA sequence
    1. Prediction is based on the need for amino acids to be in a certain position in order to achieve the desired structure
      1. Protein databases can provide a wealth of predictions
        Sequence alignment
        Domain composition
        post-translational modifications
        protein-protein interactions
        structure
        etc.
  2. Can be obtained directly by amino acid sequencing using edman degradation
  3. Or using mass spectrometry
Secondary structure
1. Alpha-helix
  1. Space-filling
  2. Backbone
  3. Sticks
  4. Ribbon
2. Experimental determination of Protein Secondary Structure by Circular Dichroism (CD)
  1. CD spectroscopy in the "far-uv" spectral region (190-250 nm) reveals secondary structure.
  2. Alpha-helix, beta-sheet, and random coil each give a characteristic shape of CD spectrum.
  3. The fraction of each secondary structure type in any protein can be calculated from its far-uv CD spectrum.
  4. This gives a % for each secondary structure element but no information on arrangement.
Tertiary structure
1. 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
2. Information on tertiary Structure from CD
  1. The CD spectrum of a protein in the "near-uv" spectral region (250-350 nm) gives some information on tertiary structure
  2. CD signals of aromatic amino acids and disulfide bonds, are sensitive to the overall tertiary structure of the protein.
  3. Their absorbance is affected by the local ‘environment’ and can be observed dynamically.
  4. Protein unfolding or ‘melting’ can be followed by CD at different temperatures.
Quaternary structure
1. Quaternary structure is the relationship between individual proteins in a multimeric complex
Folding of a peptide into a protein
Proteins are held together by different ionic interactions
1. Ionic interactions
  1. Attraction between +ve and -ve charged ions
2. Van der waals
  1. Short range weak electrical attraction and repulsion
3. Hydrogen bonds
  1. Involve a H shared between O and N atoms
Other techniques
1. X-ray crystallography
2. NMR nuclear magnetic resonance
  1. In most atoms the ‘spin’ of subatomic particles are paired against each other, such that the nucleus of the atom has no overall spin.
  2. 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.
    1. 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
  3. ‘NMR active’ nuclei (like 1H or 13C) resonate at a specific frequency in a strong magnetic field.
  4. Depending on local environment, different protons resonate at slightly different frequencies, known as a chemical shift.
  5. NMR structure determination is an iterative process, so one arrives at several possible structures which are usually represented as an ensemble.
3. Electron microscopy
  1. EM uses negative stain (shown) or vitreous ice (cryo-EM) to preserve the specimen.
    1. Image analysis is then employed to build up an average structure.
      1. 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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Protein structure (and function)

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	Created by Kristi Brogden over 10 years ago