Proteins Flashcards
1
Q
peptide bond: structure
A
- resonance hybrid
- rigid, planar, trans config. favored
- N is partially +, O is partially -
- delocalization of C-N: no rotation about bond
2
Q
Psi angle
A
C-Ca
3
Q
Phi angle
A
Ca-N
4
Q
secondary structure characteristics: all types
A
- stabilized by hydrogen bonds
- composed of regularly repeating phi and psi angles
5
Q
secondary structure: alpha helix
A
- 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
6
Q
secondary structure: beta-pleated sheets
A
- composed of beta strands
- parallel or antiparallel
- rise per residue depends on anti vs. parallel
- hydrogen bonds between neighboring chains
7
Q
beta turn
A
- proline and glycine
8
Q
tertiary protein structure: principles
A
- 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
9
Q
fibrous proteins
A
- most of polypeptide chain is organized parallel to a single axis
- mechanically strong
- insoluble in H2O
- contain one type of secondary structure per protein
10
Q
globular proteins
A
- 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
11
Q
folding forces: requirements
A
- 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
12
Q
motifs: combo types
A
- BaB loop
- aa loop
- B barrel
- aB barrel
13
Q
motif
A
- repetitive secondary structure
- clusters of secondary structure
- recognizable folding pattern with 2+ elements of secondary structure
14
Q
domain
A
- unit of tertiary structure
- stable, globular
- 3D structure remains when separated from protein
15
Q
disordered proteins
A
- 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
16
Q
alpha keratin
A
- 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
17
Q
silk fibroin
A
- 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
18
Q
collagen triple helix
A
- 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)
19
Q
protein folding: thermodynamic compromise
A
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
20
Q
protein folding: stability
A
- 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
21
Q
protein denaturation: causes
A
- detergent
- chaotropic agents (urea)
- heat
- pH change
- pressure
- organic solvents
22
Q
Tm (midpoint)
A
- 50% folded
- 50% unfolded
23
Q
cooperative folding
A
- fold to lowest-energy fold
- search is not random because direction towards native structure is thermodynamically favored
24
Q
Anfinsen’s experiment
A
- 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
25
Levinthal's Paradox
- length it would take a protein to fold if process were random
26
folding mechanism: basis
- primary structure depends on secondary and tertiary structures
27
folding mechanism: steps
1. rapid reversible formation of local secondary structure
2. aggregation of nonpolar residues (hydrophobic collapse)
3. formation of domains through cooperative aggregation of folding nuclei
- long range interactions between secondary structures and hydrophobic interactions
28
folding mechanism: thermodynamics
- free energy funnel
- unfolded: high degree of conformational entropy, low entropy of solvent
- folded: lower degree for protein, higher solvent entropy
29
chaperones and chaperonins: general facts
- protect nascent proteins from concentrated protein matrix on the cell, accelerate slow steps
- first identified in "heat shock" proteins
30
chaperones
- interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways
31
chaperonins
- elaborate complex needed for folding of certain proteins that do not fold spontaneously
32
protein purification: reasons
- find sequence and composition
- find 3D structure
33
protein purification: method
1. obtain/grow cells
2. cell lysis
3. fractionation based on solubility differences
4. dialysis
5. chromatography
34
protein purification: separation techniques
- depend on differences in physiochemical properties
35
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
36
gel filtration chromatography
separation by molecular weight
- porous material
- small proteins elute last, take longest path
37
affinity chromatography
separation by affinity
- ligand-bonding
- proteins that don't bind to ligands elute first
- add free ligand to elute proteins with affinity
38
purification monitoring
1. monitor concentration and activity
- absorbance @280 nm
- assays for activity
2. electrophoresis
- denaturing
- isoelectric focus
39
specific activity
amount of enzyme that can transform one micromole substrate/min @25 C
- # enzyme units/1 mg protein
40
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
41
primary sequence determination: small proteins (< ~50 residues)
1. reduce disulfide and alkylate cys residues
2. determine total AA composition via hydrolysis with HCl, analyse with HPLC
3. identify N-terminal residue
4. sequence w/ Edman degradation
42
primary sequence determination: reducing agents
1. BME
2. DTT
43
primary sequence determination: alkylating agent
1. iodoacetamide
44
primary sequence determination: large proteins (> ~50 residues)
1. repeat steps for small proteins
2. digest into smaller peptides suitable for Edman degradation
45
chromotrypsin
cleaves on C-side of Phe, Trp, Tyr (aromatics)
46
trypsin
cleaves on C-side of basic AA (Arg, Lys)
47
CNBR
cleaves on C-side of methionine
