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Biochem > Proteins > Flashcards

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
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2
Q

Psi angle

A

C-Ca

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3
Q

Phi angle

A

Ca-N

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4
Q

secondary structure characteristics: all types

A
  • stabilized by hydrogen bonds
  • composed of regularly repeating phi and psi angles
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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
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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
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7
Q

beta turn

A
  • proline and glycine
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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
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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
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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
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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
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12
Q

motifs: combo types

A
  • BaB loop
  • aa loop
  • B barrel
  • aB barrel
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13
Q

motif

A
  • repetitive secondary structure
  • clusters of secondary structure
  • recognizable folding pattern with 2+ elements of secondary structure
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14
Q

domain

A
  • unit of tertiary structure
  • stable, globular
  • 3D structure remains when separated from protein
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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
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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
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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
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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)
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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
Q

Levinthal’s Paradox

A
  • length it would take a protein to fold if process were random
26
Q

folding mechanism: basis

A
  • primary structure depends on secondary and tertiary structures
27
Q

folding mechanism: steps

A
  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
Q

folding mechanism: thermodynamics

A
  • free energy funnel
    • unfolded: high degree of conformational entropy, low entropy of solvent
    • folded: lower degree for protein, higher solvent entropy
29
Q

chaperones and chaperonins: general facts

A
  • protect nascent proteins from concentrated protein matrix on the cell, accelerate slow steps
  • first identified in “heat shock” proteins
30
Q

chaperones

A
  • interact with partially folded or improperly folded polypeptides, facilitating correct folding pathways
31
Q

chaperonins

A
  • elaborate complex needed for folding of certain proteins that do not fold spontaneously
32
Q

protein purification: reasons

A
  • find sequence and composition
  • find 3D structure
33
Q

protein purification: method

A
  1. obtain/grow cells
  2. cell lysis
  3. fractionation based on solubility differences
  4. dialysis
  5. chromatography
34
Q

protein purification: separation techniques

A
  • depend on differences in physiochemical properties
35
Q

ion-exchange chromatography

A

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
Q

gel filtration chromatography

A

separation by molecular weight
- porous material
- small proteins elute last, take longest path

37
Q

affinity chromatography

A

separation by affinity
- ligand-bonding
- proteins that don’t bind to ligands elute first
- add free ligand to elute proteins with affinity

38
Q

purification monitoring

A
  1. monitor concentration and activity
    - absorbance @280 nm
    - assays for activity
  2. electrophoresis
    - denaturing
    - isoelectric focus
39
Q

specific activity

A

amount of enzyme that can transform one micromole substrate/min @25 C
- # enzyme units/1 mg protein

40
Q

molecular weight vs. SDS-PAGE

A

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
Q

primary sequence determination: small proteins (< ~50 residues)

A
  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
Q

primary sequence determination: reducing agents

A
  1. BME
  2. DTT
43
Q

primary sequence determination: alkylating agent

A
  1. iodoacetamide
44
Q

primary sequence determination: large proteins (> ~50 residues)

A
  1. repeat steps for small proteins
  2. digest into smaller peptides suitable for Edman degradation
45
Q

chromotrypsin

A

cleaves on C-side of Phe, Trp, Tyr (aromatics)

46
Q

trypsin

A

cleaves on C-side of basic AA (Arg, Lys)

47
Q

CNBR

A

cleaves on C-side of methionine

Biochem (8 decks)

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