1
Q
What is the first step in protein purification from cells?
A
Cell lysis (breaking open cells) to release proteins into a crude extract.
2
Q
What method separates organelles or subcellular fractions based on size/density using centrifugal force?
A
Differential centrifugation.
3
Q
What is ‘salting out’?
A
A purification technique where high salt concentrations (e.g., ammonium sulfate) precipitate proteins by lowering their solubility.
4
Q
What is the purpose of dialysis?
A
To remove small solutes (like salts) from a protein solution by diffusion through a semipermeable membrane.
5
Q
In column chromatography, what are the two phases?
A
The stationary phase (solid porous matrix) and the mobile phase (buffered solution).
6
Q
What property does Ion-Exchange Chromatography exploit?
A
Differences in the net electric charge of proteins at a given pH.
7
Q
What is a ‘cation exchanger’?
A
A resin with bound anionic groups that binds positively charged proteins.
8
Q
What is an ‘anion exchanger’?
A
A resin with bound cationic groups that binds negatively charged proteins.
9
Q
How are proteins eluted from an ion-exchange column?
A
By changing the pH or increasing the salt concentration (salt gradient).
10
Q
In Size-Exclusion Chromatography (Gel Filtration), which proteins elute first?
A
Large proteins elute first because they cannot enter the pores of the beads and take a shorter path.
11
Q
Which proteins elute last in Size-Exclusion Chromatography?
A
Small proteins, because they enter the pores and are retarded by the labyrinthine path.
12
Q
What is Affinity Chromatography based on?
A
The specific binding affinity of a protein for a ligand (e.g., ATP) covalently attached to the column matrix.
13
Q
How is a protein eluted in Affinity Chromatography?
A
By adding a high concentration of free ligand or changing conditions (like salt/pH) to weaken binding.
14
Q
What is HPLC?
A
High-Performance Liquid Chromatography; it uses high pressure to speed up flow and improve resolution.
15
Q
What does ‘Specific Activity’ measure?
A
The purity of an enzyme (units of enzyme activity per milligram of total protein).
16
Q
What happens to Specific Activity during a successful purification?
A
It increases as unwanted proteins are removed while the enzyme of interest is retained.
17
Q
What is Electrophoresis used for?
A
To visualize and characterize proteins based on migration in an electric field (analytical, not usually preparative).
18
Q
What is the role of SDS (Sodium Dodecyl Sulfate) in SDS-PAGE?
A
It is a detergent that unfolds proteins and gives them a uniform negative charge-to-mass ratio.
19
Q
On what basis does SDS-PAGE separate proteins?
A
Almost exclusively by molecular weight (mass).
20
Q
In SDS-PAGE, do smaller or larger proteins migrate faster?
A
Smaller proteins migrate faster (found at the bottom of the gel).
21
Q
What is Isoelectric Focusing?
A
A technique that separates proteins according to their isoelectric point (pI) in a pH gradient.
22
Q
What is 2D Electrophoresis?
A
A combination of isoelectric focusing (first dimension) and SDS-PAGE (second dimension).
23
Q
What does the vertical separation in 2D electrophoresis represent?
A
Differences in molecular weight.
24
Q
What does the horizontal separation in 2D electrophoresis represent?
A
Differences in pI (isoelectric point).
25
How can you visualize proteins in a gel?
By staining with a dye like Coomassie blue.
26
What is the Primary Structure of a protein?
The sequence of amino acid residues linked by peptide bonds (and disulfide bonds).
27
Who first sequenced a protein (insulin)?
Frederick Sanger.
28
What reagent did Sanger use to label the N-terminal amino acid?
FDNB (1-fluoro-2,4-dinitrobenzene).
29
What is the Edman Degradation used for?
To sequence a protein one amino acid at a time from the N-terminus.
30
What does the protease Trypsin do?
It cleaves peptide bonds on the carboxyl side of Lysine (Lys) and Arginine (Arg).
31
What does Chymotrypsin cleave?
Peptide bonds adjacent to aromatic residues (Phe, Trp, Tyr).
32
What does Cyanogen Bromide cleave?
Peptide bonds adjacent to Methionine (Met) residues.
33
What is Mass Spectrometry (MS) used for in protein chemistry?
To determine molecular mass with high precision and sequence short peptides.
34
What is MALDI MS?
Matrix-Assisted Laser Desorption/Ionization; uses laser pulses to ionize proteins in a matrix.
35
What is ESI MS?
Electrospray Ionization; forces solution through a charged needle to create charged microdroplets.
36
What is Tandem MS (MS/MS)?
A technique using two mass filters; the first selects a peptide, the second analyzes fragments to determine sequence.
37
Can small peptides be chemically synthesized?
Yes, using the Merrifield method on a solid support.
38
In which direction does chemical peptide synthesis proceed?
From the Carboxyl terminus to the Amino terminus (opposite of biological synthesis).
39
What are Homologous proteins (Homologs)?
Members of a protein family that share a common ancestor.
40
What are Paralogs?
Homologs present in the same species (usually arising from gene duplication).
41
What are Orthologs?
Homologs found in different species.
42
What is a Consensus Sequence?
A sequence reflecting the most common amino acid at each position when related proteins are aligned.
43
What is a Signature Sequence?
A specific segment unique to a certain taxonomic group of organisms.
44
What are Conservative Substitutions?
Replacing an amino acid with one of similar chemical properties (e.g., Glu for Asp).
45
What is Horizontal Gene Transfer?
The transfer of a gene from one organism to another (rare), complicating evolutionary trees.
46
What are the four levels of protein structure?
Primary, Secondary, Tertiary, Quaternary.
47
What stabilizes the Primary structure?
Covalent peptide bonds.
48
What is a 'native' protein?
A protein in its functional, folded conformation.
49
Is the peptide bond rotatable?
No, it is rigid and planar due to partial double-bond character.
50
Which bonds in the polypeptide backbone can rotate?
The N-Cα (phi, φ) and Cα-C (psi, ψ) bonds.
51
What is a Ramachandran plot?
A plot visualizing the allowed values of φ and ψ dihedral angles for amino acid residues.
52
Why are some regions in a Ramachandran plot forbidden?
Due to steric interference (clashes) between atoms.
53
Which amino acid has the most restricted allowed regions?
Proline (due to its cyclic side chain).
54
Which amino acid is the most flexible (allowed in many regions)?
Glycine (due to its small side chain).
55
What drives protein folding thermodynamically?
The Hydrophobic Effect (entropy increase of water).
56
What role do Hydrogen bonds play in folding?
They stabilize secondary structures and select the correct native fold, though net energy gain is small.
57
What are Van der Waals interactions?
Weak attractive forces between dipoles over short distances, significant in packed protein cores.
58
Where are hydrophobic residues typically found in a globular protein?
Buried in the interior, away from water.
59
Where are charged/polar residues typically found?
On the surface of the protein.
60
What is a Salt Bridge?
An ionic interaction between oppositely charged groups (ion pair).
61
What defines Secondary Structure?
Local spatial arrangement of main-chain atoms (e.g., α helix, β sheet).
62
How many residues per turn in an α helix?
3.6 residues.
63
What is the pitch (rise per turn) of an α helix?
5.4 Å.
64
What stabilizes the α helix?
Intrachain hydrogen bonds between the carbonyl oxygen of residue n and the amide hydrogen of residue n+4.
65
Are α helices usually right-handed or left-handed?
Right-handed.
66
Which amino acids are strong 'helix formers'?
Alanine (Ala) and Leucine (Leu).
67
Which amino acids act as 'helix breakers'?
Proline (kink) and Glycine (too flexible).
68
What is the Helix Dipole?
A net dipole along the helix axis (positive at N-term, negative at C-term).
69
What stabilizes the β conformation?
Hydrogen bonds between backbone atoms of adjacent polypeptide strands.
70
What are the two types of β sheets?
Parallel and Antiparallel.
71
Which β sheet is more stable?
Antiparallel (H-bonds are linear).
72
What is a β turn?
A structure that reverses the direction of the polypeptide chain, often found on the surface.
73
How many residues are in a β turn?
Four.
74
Which amino acids are common in β turns?
Glycine (flexible) and Proline (cis conformation).
75
What is Circular Dichroism (CD) used for?
To estimate the fraction of secondary structure (α helix vs β sheet) in a protein.
76
What is Tertiary Structure?
The complete three-dimensional folding of a single polypeptide chain.
77
What distinguishes Fibrous proteins?
They usually have a single type of secondary structure and provide structural support.
78
What is the structure of α-Keratin?
A right-handed α helix twisted into a left-handed coiled coil.
79
How is α-Keratin cross-linked?
By disulfide bonds (rich in Cysteine).
80
What is the structure of Collagen?
A triple helix of three left-handed helical chains twisted into a right-handed superhelix.
81
What is the repeating sequence in Collagen?
Gly-X-Y (often Gly-Pro-4-Hyp).
82
Why is Glycine required at every third position in Collagen?
It is the only residue small enough to fit in the tight junction of the three chains.
83
What is the role of Vitamin C (Ascorbate) in collagen synthesis?
It is required for the hydroxylation of Proline to Hydroxyproline.
84
What disease results from Vitamin C deficiency?
Scurvy (weak connective tissue).
85
What is the structure of Silk Fibroin?
Antiparallel β sheets rich in Ala and Gly.
86
Why is silk flexible but not stretchy?
Flexible due to weak interactions between sheets; not stretchy because β strands are already extended.
87
What distinguishes Globular proteins?
They are compact, spherical, and contain multiple types of secondary structure.
88
What is a Motif (Fold)?
A recognizable folding pattern involving two or more elements of secondary structure (e.g., β-α-β loop).
89
What is a Domain?
An independently stable part of a polypeptide chain that can undergo movements as a single entity.
90
What is the 'Globin fold'?
A motif of eight α helices found in myoglobin and hemoglobin.
91
What is a β barrel?
A common motif formed by β-α-β loops arranged into a barrel shape.
92
What is an Intrinsically Disordered Protein (IDP)?
A protein that lacks a definable 3D structure in solution but is still functional.
93
What amino acids are IDPs rich in?
Charged residues (Lys, Arg, Glu) and Proline.
94
What functions do IDPs often serve?
Signal transduction hubs, scaffolds, or linkers.
95
What is Quaternary Structure?
The arrangement of multiple polypeptide subunits in a 3D complex.
96
How many subunits does Hemoglobin have?
Four (two α and two β chains).
97
What is an Oligomer (Multimer)?
A multisubunit protein.
98
What is a Protomer?
The repeating structural unit in a multimeric protein.
99
What is Denaturation?
Loss of 3D structure and function without breaking peptide bonds.
100
What agents cause denaturation?
Heat, extreme pH, organic solvents, urea, detergents.
101
What is Tm (Melting Temperature)?
The midpoint of the temperature range where 50% of the protein is unfolded.
102
What did the Anfinsen experiment with Ribonuclease A prove?
The amino acid sequence contains all the information required for a protein to fold into its native structure.
103
What reagents did Anfinsen use?
Urea (to denature) and Mercaptoethanol (to reduce disulfide bonds).
104
What is Levinthal's Paradox?
The observation that random trial-and-error folding would take forever, so folding must be directed.
105
What is the 'Free-Energy Funnel' model?
A model showing that conformational space narrows as a protein folds toward the thermodynamically stable native state.
106
What are Chaperones?
Proteins that assist the folding of other proteins and prevent aggregation.
107
What is the Hsp70 family?
Chaperones that bind to hydrophobic regions of unfolded proteins to prevent aggregation (heat shock proteins).
108
What are Chaperonins (e.g., GroEL/GroES)?
Large protein complexes that provide a chamber for proteins to fold in isolation.
109
What enzyme catalyzes the shuffling of disulfide bonds?
Protein Disulfide Isomerase (PDI).
110
What enzyme catalyzes the cis-trans isomerization of Proline?
Peptide Prolyl Cis-Trans Isomerase (PPI).
111
What are Amyloidoses?
Diseases caused by the accumulation of insoluble amyloid fibers from misfolded proteins.
112
What secondary structure is rich in amyloid fibers?
β sheets.
113
Which protein is associated with Alzheimer's disease?
Amyloid-β peptide.
114
What is a Prion?
A proteinaceous infectious particle that causes spongiform encephalopathies.
115
How do Prions cause disease?
A misfolded form (PrP^Sc) induces normal PrP^c proteins to misfold into the amyloid-like form.
116
What is PrP^Sc?
The scrapie (diseased) form of the prion protein, rich in β sheets.
117
What is Cystic Fibrosis caused by?
A deletion (Phe508) in the CFTR protein causing it to misfold and be degraded.
118
What is X-ray Crystallography?
A method to determine protein structure by analyzing the diffraction pattern of X-rays through a protein crystal.
119
What is the main limitation of X-ray Crystallography?
The protein must be crystallized, which is difficult for some proteins.
120
What does the X-ray diffraction pattern yield?
An electron-density map.
121
What is NMR Spectroscopy?
Nuclear Magnetic Resonance; determines structure of proteins in solution.
122
What is a key advantage of NMR over X-ray crystallography?
It captures dynamic changes and does not require crystals.
123
What is the size limit for NMR?
It is generally limited to smaller proteins.
124
What is Cryo-Electron Microscopy (Cryo-EM)?
A technique where samples are quick-frozen in vitreous ice and imaged with electron beams.
125
What is Cryo-EM best used for?
Large macromolecular complexes and integral membrane proteins that are hard to crystallize.
126
What is the PDB?
The Protein Data Bank, an archive of 3D structural data.
127
What is NOESY in NMR?
Nuclear Overhauser Effect Spectroscopy; measures distances between atoms in space.
128
What is Proteostasis?
The maintenance of the active set of cellular proteins (synthesis, folding, degradation).
129
What is a 'coiled coil'?
A motif where two or more α helices wrap around each other (found in keratin and myosin).
130
What is the length of an α helix with 80 residues?
120 Å (80 residues × 1.5 Å/residue).
131
Why are salt bridges stronger in the protein interior?
Because the dielectric constant is lower in the hydrophobic interior than in water.
132
Does the peptide bond have a dipole moment?
Yes, oxygen is partial negative, nitrogen-hydrogen is partial positive.
133
What are φ (phi) angles?
Rotation around the N-Cα bond.
134
What are ψ (psi) angles?
Rotation around the Cα-C bond.
135
Can φ and ψ be 0 degrees?
Generally no, due to steric overlap.
136
What is the typical dielectric constant (ε) of the protein interior?
About 4 (compared to 80 for water).
137
What is Ubiquitin?
A protein that tags other proteins for degradation.
138
What is the 'resolution' in X-ray crystallography?
The ability to distinguish two points; lower values (e.g., 1.5 Å) mean better detail.
139
What is a 'family' of proteins?
Proteins with significant similarity in primary/tertiary structure and function.
140
What is a 'superfamily'?
Two or more families with little sequence similarity but the same major structural motif.
141
What is 'induced fit'?
Conformational change in a protein upon ligand binding.
142
What is the repeating unit of the β sheet?
6.5 Å (parallel) or 7.0 Å (antiparallel).
143
Why is Proline called an 'imino' acid?
It has a secondary amine (nitrogen in a ring).
144
What percent of peptide bonds involving Proline are cis?
About 6% (vs <0.05% for others).
145
What is the 'hydrophobic collapse'?
The initial rapid stage of folding where hydrophobic residues cluster.
146
What is a 'molten globule'?
A compact, partially folded intermediate state.
147
Can protein structure be predicted from sequence computationally?
Yes, using algorithms like Rosetta or AlphaFold.
148
What is 'Foldit'?
A video game that crowdsources protein folding problems.
149
What is a 'designer protein'?
A protein with a novel fold or function created artificially.
150
What are 'Theozymes'?
Theoretical enzyme designs computed to test catalytic mechanisms.
BIOMG 3300
flashcards
Decks in class (4)
# Cards
