Proteins Flashcards
peptide bond: structure
- resonance hybrid
- rigid, planar, trans config. favored
- N is partially +, O is partially -
- delocalization of C-N: no rotation about bond
Psi angle
C-Ca
Phi angle
Ca-N
secondary structure characteristics: all types
- stabilized by hydrogen bonds
- composed of regularly repeating phi and psi angles
secondary structure: alpha helix
- identified in keratin
- stabilized by hydrogen bonds
- favorable pattern: hydrogen bonds parallel to helix axis so all N-H are oriented in the same direction
- angles allowed
secondary structure: beta-pleated sheets
- composed of beta strands
- parallel or antiparallel
- rise per residue depends on anti vs. parallel
- hydrogen bonds between neighboring chains
beta turn
- proline and glycine
tertiary protein structure: principles
- secondary structures form whenever possible due to large number of hydrogen bonds
- helices and sheets pack close together
- backbone links are short and direct
- fold to make stable structures
- minimize solvent contact, make hydrogen bonds
fibrous proteins
- most of polypeptide chain is organized parallel to a single axis
- mechanically strong
- insoluble in H2O
- contain one type of secondary structure per protein
globular proteins
- hydrophobic residues face interior and interact with each other
- polar residues face outside and interact with solvent
- internal hydrogen bonding is maximized
- close packing of residues, but ratio of van der waals volume to total volume is 0.72-0.77 so empty space exists in form of small cavities
folding forces: requirements
- peptide chain must satisfy constraints inherent in its own structure
- right handed twist
- must fold to bury hydrophobic side chains, minimizing contact with water
- substantial amounts of helices &/or sheets in core
motifs: combo types
- BaB loop
- aa loop
- B barrel
- aB barrel
motif
- repetitive secondary structure
- clusters of secondary structure
- recognizable folding pattern with 2+ elements of secondary structure
domain
- unit of tertiary structure
- stable, globular
- 3D structure remains when separated from protein
disordered proteins
- contain segments lacking definable structure
- composed of amino acids whose higher concentration forces less defined structure
- lys, arg, glu, pro
- can conform to many diff. proteins, facilitating different partner proteins
alpha keratin
- found in hair, nails, claws, horns, beaks
- right handed helix with 5.1 Å
- stabilized by intrachain hydrogen bonds
- 7 residue heptad repeats
(a-b-c-d-e-f-g)n
where a and d are nonpolar to promote association of helices - coiled coil forms left hand twist
silk fibroin
- nests, webs, egg sacs
- form B-sheets
- antiparallel stabilized by interchain hydrogen bonds and london dispersion forces
- alternating sequence
- Gly-Ala/Ser
- glycines on one side, alanine/serine on other side creates meshing effect
collagen triple helix
- tendons
- basic unit: tropocollagen
- 1/3 is glycine
- proline content is unusually high
- 30% are pro or hypro
- no disulfide
- 2.9 Å with 3.3 residues per turn
- stabilized interchain hydrogen bonds (N-H groups of gly and C=O in adjacent strand)
protein folding: thermodynamic compromise
unfolded (denatured)
- high conformational energy
- favorable AA-H2O bonds but unfavorable H2O ordering
folded (native)
- low conformational energy
- favorable stability between noncovalent (hydrogen bonds, ionic interactions, hydrophobic effect), and disulfide forces
protein folding: stability
- attributed per AA is 0.4 KJ/mol
- marginally stable due to flexibility and motion
- flexibility is essential for function
- ligand bonding
- enzyme catalysis
- enzyme regulation
- depends on 3D structure
- loss of structural integrity with loss of activity = denaturation
protein denaturation: causes
- detergent
- chaotropic agents (urea)
- heat
- pH change
- pressure
- organic solvents
Tm (midpoint)
- 50% folded
- 50% unfolded
cooperative folding
- fold to lowest-energy fold
- search is not random because direction towards native structure is thermodynamically favored
Anfinsen’s experiment
- ribonuclease, which is rich in disulfide bonds
- BME thiol and urea used to reduce
- unfolded state
- inactive, disulfide cross-links reduced to cys residues
- removal of BME and urea
- new catalytically active state, disulfide links reformed
- enough info in primary sequence to give rise to tertiary fold/native conformation
Levinthal’s Paradox
- length it would take a protein to fold if process were random
folding mechanism: basis
- primary structure depends on secondary and tertiary structures
folding mechanism: steps
- rapid reversible formation of local secondary structure
- aggregation of nonpolar residues (hydrophobic collapse)
- formation of domains through cooperative aggregation of folding nuclei
- long range interactions between secondary structures and hydrophobic interactions
folding mechanism: thermodynamics
- free energy funnel
- unfolded: high degree of conformational entropy, low entropy of solvent
- folded: lower degree for protein, higher solvent entropy
chaperones and chaperonins: general facts
- protect nascent proteins from concentrated protein matrix on the cell, accelerate slow steps
- first identified in “heat shock” proteins
chaperones
- interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways
chaperonins
- elaborate complex needed for folding of certain proteins that do not fold spontaneously
protein purification: reasons
- find sequence and composition
- find 3D structure
protein purification: method
- obtain/grow cells
- cell lysis
- fractionation based on solubility differences
- dialysis
- chromatography
protein purification: separation techniques
- depend on differences in physiochemical properties
ion-exchange chromatography
separation by charge
- resin is charged
- cation exchange: matrix is carboxymethyl, negative charge interacts with cations
- anion exchange: matrix is diethylaminoethyl, positive charge interacts with anions
gel filtration chromatography
separation by molecular weight
- porous material
- small proteins elute last, take longest path
affinity chromatography
separation by affinity
- ligand-bonding
- proteins that don’t bind to ligands elute first
- add free ligand to elute proteins with affinity
purification monitoring
- monitor concentration and activity
- absorbance @280 nm
- assays for activity - electrophoresis
- denaturing
- isoelectric focus
specific activity
amount of enzyme that can transform one micromole substrate/min @25 C
- # enzyme units/1 mg protein
molecular weight vs. SDS-PAGE
PAGE: polyacrylamide gel electrophoresis
SDS: sodium dodecyl sulfate
- 1 SDS/2 AA
- SDS binds and unfolds all proteins, gives uniformly negative charge
- smaller proteins travel faster
primary sequence determination: small proteins (< ~50 residues)
- reduce disulfide and alkylate cys residues
- determine total AA composition via hydrolysis with HCl, analyse with HPLC
- identify N-terminal residue
- sequence w/ Edman degradation
primary sequence determination: reducing agents
- BME
- DTT
primary sequence determination: alkylating agent
- iodoacetamide
primary sequence determination: large proteins (> ~50 residues)
- repeat steps for small proteins
- digest into smaller peptides suitable for Edman degradation
chromotrypsin
cleaves on C-side of Phe, Trp, Tyr (aromatics)
trypsin
cleaves on C-side of basic AA (Arg, Lys)
CNBR
cleaves on C-side of methionine
