Module 1 Section 4 Flashcards
Nested Protein levels
the four protein levels exist in a manner that one level higher is completely comprised of level below
Primary structure
-linear sequence of AA, read from N-term to C-term
Peptide backbone constraints
- alpha carbons separated by 3 covalent bonds
- C-N is the peptide bond
- the resonance in the backbone results in planar structure, partial double bond character
- no rotation around peptide bond
- N-C(alpha) angle is phi
- C(alpha)-C angle is psi
Ramachandran Plot
represents the allowed phi and psi for each AA
- glycine has larger range of allowed values (small R)
- proline is greatly restricted
- easily allowed conformations are darker on the plot
Secondary structure
- H-bonds form between polar atoms in backbone chain
- alpha helix=10-15 residues long
- beta sheet =3-10 residues
Alpha-helix
- most stable arrangement of backbone
- 3.6 AA per turn
- one turn is 5.4 angstroms long
- R groups protrude from helix
B-sheet
- second most stable backbone arrangement
- H-bonds between backbone amide and carbonyl groups
- can accommodate large aromatic residues
B-sheet orientation
- can run parallel or antiparallel orientations
- opposite N-term to C-term directions strands are antiparallel
B-turns
- reversal in direction with 4 AA residues
1. backbone carbonyl O of 1st bonds to amide H of 4th - no inter residue bonds between 2nd and 3rd residues
- usually Pro position 2, Gly position 3
Gamma turns
- less common than B-turn
- only 3 AA
- 1st+3rd residues form H-bond
- 2nd not involved
Tertiary structure
3-D orientation of all secondary structures
-main overall shapes: fibrous, globular
Globular protein
-water soluble, spherical
Fibrous proteins
- elongated shape, play structural role
- ie. keratin, collagen
quaternary structure
- the connection between 2+ polypeptide chains
- protein of 2 subunits called dimer
Multisubunit composition
- avoids problem of folding one large polypeptide (ie. one domain not folding properly renders entire protein useless)
- if one subunit misfolds, is excluded from oligomer
- if one subunit denatures, is replaced by another subunit
Denaturation
loss of biological activity
Oligomer/multimer
protein with multiple polypeptide chains
subunits/protomers
individual polypeptide chains
homooligomer
oligomer with identical subunits
heterooligomer
oligomer with non-identical subunits
Protein folding
- some proteins require assistance to fold
- structure always in flux, vibrating rapidly
Regular + Misfolded proteins
- nature state of protein selected at AA level, which is the lowest energy state
- if states achieve lower free energy than functional protein conformation, will lead to irreversible aggregations
Steps of protein purification
- Cell lysis
- Centrifugation
- Fractionation
- Protein detection
Cell lysis methods
- Detergent (compromise membrane integrity)
- Shear force (apply high freq sonic waves [sonification])
- low ionic salt (cells osmotically absorb water + burst)
- Pressure changes (high pressure breaks cells)
About centrifugation of cell lysate
- uses sedimentation principle (denser particles sediment at bottom)
- pellet at bottom, supernatant at top
- differential centrifugation is at different speeds
Fractionation by Column Chromatography
- mixture dissolved in mobile phase
- mobile phase passes through stationary phase
- stationary phase causes components of mobile phase to move at different speeds (ie. size, charge)
Steps of column chromatography
- protein mixture applied to column with resin or matrix
- buffer passed through column to wash away proteins that don’t bind with matrix
- another buffer applied that causes bound proteins to dissociate from matrix (elution)
- eluted proteins collected in fraction collector
Types of column chromatography
- Ion exchange
- Size-exclusion
- Affinity
About Ion exchange chromatography
- resin contains anion/cation exchange resin bonds anions/cations respectively
- elution due to increasing salt solution concentration
- pI used for each protein, character depends on the pH of solution dissolving proteins
About size exclusion chromatography
- matrix has pores of specific size, proteins smaller than pore can enter
- proteins that enter pores take longer to elute
- large proteins elute fastest
About affinity chromatography
- uses fact that many proteins specifically bind other mlc/ligands as part of their function
- column can have ligand covalently attached to matrix
SDS-PAGE steps
- used for protein detection
1. treated samples loaded into wells @ top of gel, electric field applied to gel (SDS highly -ve so proteins move towards +ve cathode @ bottom)
2. proteins migrate at diff rates accoording to relative molecular mass (smaller=faster)
3. Gel removed from between glass, soaked in acidic buffer to ‘fix’ proteins (gel treated w dye that selectively binds to proteins)
