ME01 - Amino Acids and Peptides Flashcards
(172 cards)
1
Q
Difference of alpha helix and B sheets
A
ALPHA HELIX
Polypeptide backbone twisted
R groups face outward
Stability due to H bonds between oxygen of the peptide bond carbonyl and H atom of the peptide bond nitrogen
Peptide backbone is highly extended
AA residues form zigzag or pleated pattern
R groups point on opposite direction
2
Q
What provides the thermodynamic driving force of alpha helices
A
The maximum number of H bonds coupled with Van der Waals forces
3
Q
Proline and alpha helix
A
Proline lack a Hydrogen atom and could only be stable accommodated within the first turn
4
Q
Glycine and alpha helix
A
Glycine induces a bend
5
Q
Types of Beta-Sheet – Parallel and Adjacent
A
Parallel - adjacent segments proceed in the same amino to carboxyl
Antiparallel - opposite directions
6
Q
Isolating specified protein for determination of physical and functional protein properties
A
Protein Purification
7
Q
Approaches for Protein Purification
A
pH (isoelectric precipitation)
Polarity (ethanol or acetone precipitation)
Salt concentration (ammonium sulfate salting)
8
Q
Optimal separation achieved by manipulation of the composition of the 2 phases
A
CHROMATOGRAPHY
Mobile and Stationary Phase
9
Q
Proteins interacting more strongly with the stationary phase are retained longer
A
True.
10
Q
Conformation vs Configuration
A
CONFORMATION
- Spatial relationship between atoms
- Interconversion does not need bond rupture
- Retention of configuration
- Rotation about single bonds
CONFIGURATION
- Geometric relationship between atoms
- Interconversion needs breaking of covalent bonds
- L-amino acids vs D-amino acids
11
Q
How do you extract soluble proteins
A
Extract using aqueous solutions at physiologic pH and ionic strength
12
Q
Type of proteins requires detergent solutions
A
Integral membrane proteins
13
Q
What are the stationary and mobile phase of Column Chromatography?
A
Stationary phase: column containing spherical beads or modified cellulose, acrylamide or silica whose surface has been coated with chemical functional groups
Mobile phase: Liquid type. Percolated and goes through.
Small portions of the mobile phase/eluant are collected
14
Q
Separation of mixture in columns based on partition of a solute between 2 solvents one of which is immobilized by the substance in the column or paper
A
Partition Chromatography
15
Q
Also known as gel filtration
A
Size Exclusion Chromatography
16
Q
What type of Chromatography uses Stoke radius
A
Size Exclusion Chromatography
17
Q
Separates proteins based on their Stoke radius
A
Size Exclusion Chromatography
18
Q
Relation of Stoke Radius with Size Exclusion Chromatography
A
Proteins with large Stoke radii remain in the eluent and emerge before the proteins that have a smaller Stokes radii and are able the porous beads
19
Q
Protein mixture is applied to a column under conditions where protein of interest associates with the stationary phase
A
Absorption Chromatography
Partition coefficient is unity = 1
20
Q
How are proteins sequentially released and what to use for Size Exclusion Chromatography
A
By disrupting the forces that stabilize the protein-stationary phase complex.
A gradient of increasing salt concentration.
21
Q
Why is the mobile phase in Size Exclusion Chromatography being gradually altered?
A
So that molecules are selectively released in descending order of their affinity.
22
Q
Method of protein selection that employs incompressible silica or alumina microbeads as stationary phase and pressures of up to a few thousand psi
A
High_Pressure Liquid Chromatography
23
Q
HPLC and incompressible matrices
A
Incompressible matrices permit both high flow rate and enhanced resolution
24
Q
Complex mixtures of lipids or peptides uses what type of chromatography
A
High Pressure Liquid Chromatography
25
Exploits a hydrophobic stationary phase or aliphatic polymers 3-18 carbon atoms in length
Reversed phase HPLC
26
How are peptide mixtures eluted using HPLC
By using a gradient of a weak-miscible organic solvent such as acetonitrite or methanol
27
Proteins interact with the stationary phase via charge-charge interactions
Ion Exchange Chromatography
28
Concept of Ion Exchange Chromatography
Proteins with (+) charge at a given pH adhere beads with negatively charged functional groups such as carboxylates or sulfates (cation exchangers)
29
Why is it called "Ion-Exchange Chromatography"
"Ion-exchange" because proteins compete with monovalent ions for binding
30
Negatively charged proteins bind to diethylaminoethyl (DEAE) cellulose via replacing the Cl- or CH3COO-. What type of Chromatography is used?
Ion Exchange Chromatography
31
Proteins elute in inverse order of strength of their interactions with stationary phase?
Ion Exchange Chromatography
32
How do you achieve sequential elution in Ion Exchange Chromatography?
pH manipulation. Since it involves ions and charges
33
Separates via tendency to associate with a stationary phase matrix coated with hydrophobic groups phenyl Sepharose, octyl Sepharose
Hydrophobic Interaction Chromatography
34
Relation of Ionic Strength with Hydrophobic Interaction Chromatography
High Ionic Strength enhances adherance of proteins with exposed hydrophobic surfaces to the matrix via hydrophobic interactions
35
Polarity and Hydrophobic Interaction Chromatography
Polarity of mobile phase is decreased gradually lowering salt interaction
36
What is added to decrease the polarity and weaken hydrophobic interactions?
Ethanol or Glycerol
37
Exploits high selectivity of most proteins for their ligands. What type of Chromatography is used?
Affinity Chromatography
38
How are enzymes purified in Affinity Chromatography?
By using immobilized substrates, products, coenzymes or inhibitors
39
How are bound proteins eluted in Affinity Chromatography?
Either by competition with soluble ligand or less selectively by disrupting protein-ligand interactions using urea, guanidine HCl, mildly acidic pH and high salt concentration
40
Stationary phase of Affinity Chromatography
Contain ligands such as NAD+ or ATP analogs
41
Most powerful and widely applicable affinity matrices are used for -- in Affinity Chromatography?
Recombinant protein purifications
42
Recombinant Protein Purification is involved and what type of Chromatography?
Affinity Chromatography
43
Explain Recombinant Protein Purifications
Uses an Ni2+ matrix that binds proteins with an attached polyhistidine tag and a glutathione matrix that binds a recombinant protein linked to glutathione S-transferase
44
Most widely used method for determining protein purity
Polyacrylamide Gel Electrophoresis
45
What is used in Polyacrylamide Gel Electrophoresis?
Sodium Dodecyl Sulfate
46
Concept of Polyacrylamide Gel Electrophoresis
Electrophoresis separates charged biomolecules based on the rates at which they migrate in an applied electrical field
47
What is polymerized and cross-linked to form porous matrix
Acrylamide
48
Denatures and binds to protein at a ration of one molecule per 2 peptide bonds in Polyacrylamide Gel Electrophoresis
SDS - Sodium Dodecyl Sulfate
49
What is used to to reduce or break disulfide bonds in Polyacrylamide Gel Electrophoresis
2-Mercatoethanol or Dithiothreitol
50
What determines the rate of migration in Polyacrylamide Gel Electrophoresis
The physical resistance encountered by the polypeptide
51
Polypeptides and Polyacrylamide Gel Electrophoresis
Polypeptides separate based on their relative molecular mass (Mr)
52
What stain is used when individual polypeptides are trapped in the gel in the Polyacrylamide Gel Electrophoresis
Coomassie Blue
53
Ionic buffers and an applied electric field are used to generate pH gradient within a polyacrylamide matrix
Isoelectric Focusing
54
Explain Isoelectric Focusing
Applied proteins migrate until they reach the region of the matrix where the pH at which a molecule's net charge is 0.
55
The one who determined the amino acid sequence of Insulin
Frederick Sanger
56
Separated both chains by reducing disulfide bonds and cleaved with trypsin, chymotrypsin and pepsin.
Sanger Amino Acid Sequencing
57
Who introduced Edman's reagent?
Pehr Edman. Other term for Edman's reagent is phenylisothiocyanate
58
Phenylthiohydantoin (PTH) derivative can be removed under mild conditions to generate a new amino terminal residue
EDMAN Amino Acid Sequencing
59
What is necessary to circumvent post translational modifications and to render a proteins alpha-amino group unreactive to Edman's reagent
Cleavage
60
Steps for EDMAN Amino Acid Sequencing
Cleavage
Peptides are purified by reversed phase HPLC
Sequencing
61
Provides a partial amino acid sequence
EDMAN Amino Acid Sequencing
62
Oligonucleotide primers modeled are used to ID the gene and amplify it using PCR
Hybrid Approach
63
STEPS in Hybrid Approach
ID the gene
| Oligonucleotide sequence is determined to infer the primary structure of the encoded polypeptide
64
Enhances the speed and efficiency of primary structure analysis
Hybrid Approach
65
Only a few segments of the primary structure should be determined by what approach
Edman's approach
66
Order of which amino acids are added in DNA sequencing
```
Proteolytic processing
Methylation
Glycosylation
Phosphorylation
Proline and Lysine Hydroxylation
Disulfide bond formation
```
67
Replaced Edman's sequencing
Mass Spectrometry
68
How is post translational modification identified in Mass Spectrometry
Via Mass increments
69
Bias in Mass Spectrometry
Discriminates molecules based solely on mass
70
A sample in vacuum is vaporized under conditions where protonation can occur, imparting positive charge
Mass Spectrometry
71
The force of ions with identical net charge is proportionate to their mass in Mass Spectrometry, T or F.
True.
72
Molecules with identical net charge have velocities inversely proportional to their mass. T of F
True.
73
Type of Mass Spec suited for large protein masses.
Time-of-flight mass specs
74
Types of Mass Specs that permitted determination of large polypeptide masses
Matrix-assisted laser-desorption (MALDI)
| Electrospray dispersion
75
Peptides inside the mass spec are broken down into smaller units by collision with natural helium
Collision-induced dissociation
76
Peptide bond are more labile than carbon-carbon bond
True
77
Molecular mass of each amino acid is unique except for
Leucine and Isoleucine
78
Used to screen blood samples from newborn (Newborn Screening)
Tandem Mass Spec
79
Used for abnormalities in metabolites (phenylketonuria, ethylmalonic encephalopathy and glutaric acidemia type I)
Tandem Mass Spec
80
Genomics uses what approach
Hybrid Approach
81
How many residues are needed to identify the correct open reading frame (ORF)
4-5 residues
82
What is Peptide mass profiling
A peptide is digested and introduced into the mass spec
83
A computer program is used to find an ORF whose predicted protein product would produce a set of peptides whose masses match the ones in the mass spectrometry
Genomics
84
Aimd to id the entire complement of proteins elaborated by a cell under diverse conditions
Proteomics
85
Proteins whose appearance/disappearance are associated with a specific physiologic condition or disease
Proteomics
86
Utilizes robotic automation
Proteomics
87
Alternative and complementary approach that utilizes mRNA that encodes proteins
DNA Chips
88
Gene arrays are more sensitive
True
89
Compact, Spherical with axial ratios
Globular Proteins
90
Axial Proteins >10
Fibrous Proteins
91
Lipid protein
Lipoproteins
92
Carbohydrate protein
Glycoprotein
93
Tightly-associated metal ions
Metalloproteins
94
Structure - Amino Acid Sequence
Primary Structure
95
Structure - Folding of 3-30 amino acid residues forming geometrically ordered units
Secondary Structure
96
Structure - Assembly of secondary structure into larger functional units
Tertiary Structure
97
What is the Quarternary Structure
Polypeptide unts of oligomeric proteins and spatial arrangements
98
Provides both molecular fingerprint for ID and information to determine the clone the gene that encodes it
Primary Structure
99
Acid strength
pKa
100
Net charge of pKa
Sum of all the (+) and (-) charged groups present - depends on the pKa values
101
Polarity and Environment
Polar env't favors charged form
| Nonpolar env't favors uncharged
102
pH midway between pKa values of an isoelectric species
pI
103
pI is isoelectric
Molecule has equal number of (+) and (-) charges
104
Amino acids do not absorb visible light and are colorless. T or F
True
105
Proteins that absorb high wavelength
Tyrosine, Phenylalanin, Tryptophan
106
Smallest aa found in peptide bend
Glycine
107
Hydrophobic Proteins
```
MVP TTAIL
Methionine
Valine
Phenylalanine
Tryptophan
Tyrosine
Alanine
Isoleucine
Leucine
```
108
Amino acids with BASIC side chains
HArgLys
Histidine
Arginine
Lysine
109
Amino acids with ACIDIC side chain
Aspartic Acid
| Glutamic Acid
110
Amino Acids with polar but uncharged side chains
```
Ser Gln, Thrash'n
Serine
Glutamine
Threonine
Asparagine
```
111
Amino Acids with Special Cases
Cysteine
Glycine
Proline
112
Where is free rotation possible on Secondary Structure
1. Ca to Co
| 2. Ca to N
113
Why is partial double bond character of Co to a-N requires carbonyl carbon
Prevent rotation
114
Carbonyl Oxygen and a-Nitrogen remain coplanar
Prevent rotation
115
Angle about the Ca- N
Phi (o)
116
Angle about the Ca-Co
Psi (y)
117
Most o(phi) and y(psi) are not allowed due to steric hindrances except
Glycine
118
Why is Proline restricted in the secondary structure
Due to absence of Ca -N
119
Most common secondary structure
a-helix and B-sheets
120
Joining two units of secondary structure
Loops and Bends
121
4 aa residues - Carbonyl oxygen of the first residue forms a hydrogen bond with the amino-group hydrogen of the fourth residue
B turn
122
Examples for B turn
Proline and Glycine
123
Regions that contain residues beyond the minimum number necessary to connect adjacent region of secondary structure
Loops
124
Serve key biologic function
Loops
125
Describe LOOPS
Lack apparent structural regularity
| In enzymes, bridge domains which bind substrates and contain aa residues that participates in catalysis
126
Stabilizes loops
H bonding, Salt bridges and Hydrophobic forces
127
Provide oligonucleotide-binding portion of DNA-binding proteins such as repressors and transcription factors
Helix-Loop-Helix Binding
128
Intermediate between 2 and 3 structure
Supersecondary structures
129
Accessible sites for antibody recognition and binding
Epitopes
130
Where do loop and bends reside
On protein surfaces
131
Not all proteins are ordered
True
132
Often at extreme amino or carboxyl terminal
Disordered regions
133
Refers to the entire 3D conformation of polypeptide
Tertiary Structure
134
Helices, sheets, bends, turns and loops
3-D spatial behavior
135
Protein structure sufficient to perform a particular chemical or physical task such as substrate or ligand binding
Domains
136
Functions of Tertiary Structure
Anchor protein to a membrane
| Interact with a regulatory molecule modulating its function
137
Examples of Single Domains
Triose phosphate isomerase
138
Examples of Two Domains
Protein Kinases
139
What is rich in B sheets and binds ATP
Amino terminus
140
What is rich in a-helices and binds peptide/protein structure
Caboxyl terminus
141
Where do the groups that catalyze phosphoryl transfer reside in?
Reside in a loop positioned at the interface of the 2 domains
142
Defines the polypeptide composition of a protein and the spatial relationships between subunits or protomers
Quaternary Structure
143
Single polypeptide Chain
Monomer
144
Two polypeptide Chain
Dimer
145
2 copies of the same polypeptide
Homodimer
146
2 copies of different polypeptide
Heterodimer
147
Elements of quarternary structure in terms of genes
A2B2Y2
148
Non covalent factors stabilizing tertiary and quarternary structure
Hydrophobic interactions drive hydrophobi aa side chains into interior
Hydrogen bonds
Salt bridges
149
Covalent factors stabilizing tertiary and quarternary structure
80-120 kcal/mol per bond
| Disulfide bridges
150
Steps for Xray Crystallography
1. Protein is precipitated to form crystals
2. Crystals are mounted into quartz capillaries and irradiated with monochromatic x rays of 0.15 nm to confirm protein nature
3. Crystals are frozen in liquid nitrogen
4. Diffraction patterns are recorded.
5. Data is analyzed and summates wave functions
151
Measures the absorbance of radio frequency electromagentic energy by certain atomic model
Nuclear Magnetic Resonance Spectroscopy
152
A function of both the functional group within which it resides and the proximity of other NMR-active nuclei
Frequency or chemical shift in NMR Spectroscopy
153
Analyzes proteins in aqueous solution therefore conformation accompanying ligand binding or catalysis is possible
NMR Spectroscopy
154
Less than or equal to 30 kDA in size is analyzable uses this kind of Spec
NMR Spectroscopy
155
Computer technology determining the 3D protein structure
Molecular modeling
156
3-D structure of protein is used as a template to build a model of the probable structure of a related protein
Homology modeling
157
Cell organelle that participates in folding process
Ribosomes
158
Extreme high concentrations of protein in cells
Affects kinetic of protein folding
159
Protein conformation vs Protein folding
Protein conformation is energetically favored.
| Protein folding occurs in a stepwise fashion.
160
Stage in Protein Folding : Newly synthesized polypeptide emerges from the ribosomes and short segments fold into secondary structural units
First Stage
161
Stage in Protein Folding where forces that drive the hydrophobic regions into the interior of the protein away from solvent drive the partially folded polypeptide into a molten globule
Second stage
162
Proteins that undergo spontaneous folding
Denatured proteins (treated with chaotropic agents like detergents)
163
Proteins that assist in folding of over half of mammalian proteins
Chaperones
164
Bind to short hydrophobic aa shielding them from a polar solvent
Hsp 70
165
Differ in sequence and acts later in folding together with Hsp 70
Hsp 60
166
Provides a donut-shaped central cavity which shelters polypeptide
Hsp 60
167
Diseases of Protein Folding
Prion Diseases, Alzheimers disease, Beta-Thalassemia
168
Configuration of PrPc normal protein into an abnormal PrPSc
Prion Disease
169
Feature of Prion Disease
Beta-pleated sheet stacking
| Amyloid plaques which then cause CJD, Kuru, MCD, BSE, Scrapie and other prion-related diseases
170
Composed of misfolded tangles in proteins
Azheimer's Disease
171
What is Tau in Alzheimer's Disease
Tau is misfolded tangle in protein which is responsible for maintaing microtubule organization of the nerve cells
172
Causes of Alzheimer's Disease
Plaques and Misfolded Tangles
