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Proteins (polypeptides) - Coggle Diagram

- - - - Aliphatic side chains
        
        Glycine, Alanine, Valine, Leucine, and Isoleucine
    - - Bonding about the \(\alpha\)-carbon is tetrahedral
      - henever a carbon atom has four different substituents attached to it, forming an asymmetric molecule, the carbon is said to be chiral, or a center of chirality or a stereocenter (or asymmetric carbon)
        
        Most (\alpha\)-amino acids contain an asymmetric \(\alpha\)-carbon and thus have L and D enantiomers
        
        Only L-enantiomers are found in proteins
        
        All amino acids except glycine can exist in D and L forms, since in each case the \(\alpha\)-carbon is chiral. Glycine is the sola exception, because two of the groups on the \(\alpha\)-carbon are the same (H), eliminating the chirality
- - - - Separating or purifying proteins by placing them in a gel matrix and then observing protein mobility in the presence of an electrical field
      - SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis)
        
        Most common separation technique
        
        Separates proteins mainly on the basis of molecular weight as opposed to charge or folding
    - - Molecules are separated based on their difference in electric charge
      - Usually performed in a gel and takes advantage of the face that a molecule´s charge changes with the pH of its surroundings
    - - High-performance liquid chromatography
        
        Useful in combination with gel-based approaches
      - Thin-layer chromatography
        
        Useful in combination with gel-based approaches
      - Chromatography methods are commonly used methods used for purification, identification and quantification of protein mixtures.
    - - Commonly used for pre-column size and polydispersity management
    - - Powerful, gel based method used to analyze complex samples in the interest of characterizing the full range of proteins in the sample (not just a few specific proteins)
  - - - Separation by size
      - Transfer to a solid support
      - Marking target protein
        
        Using a proper primary and secondary antibody to visualise
    - - Most common method
      - Used to detect specific proteins in a given sample of tissue homogenate or extract
      - The sample of proteins is first electrophoresed by SDS-PAGE to separate the proteins based on molecular weight
        
        The proteins are then transferred to a membrane where they are probed using antibodies specific to the target protein
  - - - A method of sequencing amino acids in a peptide
      - The amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues
    - - Analytical technique
      - Measures the mass-to-charge ratio of charged particles for determining masses of particles and the elemental composition of a sample of molecules as well as for elucidating the chemical structure of molecules such as peptides
      - Important method for mass determination and characterization of proteins
      - Peptide mass fingerprinting
        
        uses masses of proteolytic peptides as input to a search of a database of predicted masses that arise from digestion of a list of known proteins
        
        Main advantage: Doesn't depend of protein sequencing for protein identification
        
        Limitation: Requires the database to have the protein which is already characterized on another organism
      - De novo peptide sequencing
        
        Is performed without any prior knowledge of the amino acid sequence
        
        Advantage: Do not need the protein database
        
        Uses computational approaches to deduce the sequence of peptide directly from the experimental MS/MS spectra
        
        Can be used for: unsequenced organisms, antibodies, peptides with posttranslational modifications (PTMs) and endogenous peptides
- - - - Primary use: Size measurements
      - Proteins have a very consistent composition and fold into tight structures and the hydrodynamic size therefore relates predictably with molecular weight
      - The most sensitive technique for detecting small quantities of aggregates in preparations
    - - Any increase in size of a protein will most likely result in aggregate formation
      - The earliest stages of denaturation will result in changes in the mean hydrodynamic size
    - - Often highly purified, many protein sample should be applicable for batch measurements of molecular weight using SLS, as long as the concentrations are accurately known
      - By measuring the amount light scattered at different concentrations of sample, the molecular weight can be calculated by creating a Debye plot
        
        The slope of the line is 2x the 2nd virial coefficient (a measure of molecular interaction within a solution) so this technique can be useful for studying crystallisation conditions
        
        A strong positive value indicates good solubility while a strongly negative value indicates a propensity to aggregate
    - - A significant number of the functional groups on amino acids can be charged and any combination of these may be in their charged or uncharged states in the protein
        
        This will change depending on the conditions in the local environment and it is important to note that zeta-potential can be different from the calculated net charge based on the likely state of the charge residues in the molecule
      - Charge is of particular interest to protein chemists
      - Zeta potential should be able to compete with iso-electric focusing
        
        A sit allows the protein to be kept in conditions far nearer to its native state
      - Difficulty with zeta potential: Proteins are subject to being denatured by the applied electric field
      - Zeta potential is a measure of the repulsive forces between molecules in solution
        
        Has conventionally been used as a primary indicator of stability of a sample preparation
        
        High zeta potential - high intermolecular repulsive force: a drug or protein preparation can be expected to be stable for longer periods than a similar preparation with low zeta potential
  - - - At known concentrations, measured with a refractive index or UV detector, molecular weight can be related directly to the amount light scattered by a molecule
  - - - Optically chiral molecules will preferentially absorb one direction of the circularly polarized light
        
        The difference in absorption of the left and right circularly polarized light can be measured and quantified
    - - Used to determine aspects of protein secondary structure
    - - Used to study the structure of small organic molecules, proteins and DNS
    - - Investigates charge transfer transitions in metal-protein complexes