Weeks 1 and 2 Flashcards
(107 cards)
1
Q
Alanine
A
Ala, A
2
Q
Valine
A
Val, V
3
Q
Leucine
A
Leu, L
4
Q
Isoleucine
A
Ile, I
5
Q
Methionine
A
Met, M
6
Q
Phenylalanine
A
Phe, F
7
Q
Glycine
A
Gly, G
8
Q
Proline
A
Pro, P
9
Q
Cysteine
A
Cys, C
10
Q
tryptophan
A
Trp, W
11
Q
Tyrosine
A
Tyr, Y
12
Q
Serine
A
Ser, S
13
Q
Threonine
A
Thr, T
14
Q
Glutamine
A
Gln, Q
15
Q
Asparagine
A
Asn, N
16
Q
Lysine
A
Lys, K
17
Q
Histidine
A
His, H
18
Q
Arginine
A
Arg, R
19
Q
Aspartate
A
Asp, D
20
Q
Glutamate
A
Glu, E
21
Q
HH equation
A
Ph= Pka + log [deprotinated]/[protinated]
22
Q
Thin-layer chromatography (TLC)
A
Polar molecules bind to the silica gel and take longer to enter the mobile phase (very polar= very low Rf)
23
Q
Column chromatography
A
The same concept as TLC, but they are measured on their elution volume
24
Q
Ion exchange chromatography
A
Separation on the basis of the charge
Cation exchange resins contain negative groups (bind cations)
Anion exchange binds negative ions
Always compared to a common standard
The volume of the buffer needed to move the amino acid from the top to the bottom of the column is the elution volume
25
Metal affinity chromatography
The column is made up of resin containing Ni2+ which His binds to
His tags are added to the N or C terminal
The high degree of purification in one step
26
Gel filtration chromatography
Beads of polymeric gel (water-filled pores)
Protein molecules can only enter the pores if they fit, the bigger ones sink to the bottom faster. Can be used to measure the molar mass of proteins
Elution volume is a linear function of log molar mass (negative slope)
27
Ultracentrifugation
Protein is placed in a centrifuge and spun really fast, the molecule sediments at a rate that is dependent on the shape and size. By measuring the sedimentation, it is possible to measure the shape and size.
28
Electrophoresis
Separation based on the movement of charged molecules in an electric field
Rate of movement depends on the shape, size and charge
Protein can be treated with Sodium dodecyl sulphate (SDS). Causes the protein molecule to extend and gives a uniform size per unit charge.
29
Isoelectric focusing
High pH, protein is deprotonated and moves towards the + electrode
As it passes through a gradient of decreasing pH, the -ve charge decreases. When the net charge is zero, the protein stops moving (this is the isoelectric point)
Each protein has a different isoelectric point
30
Two-dimensional gels
Combines of isoelectric focusing and SDS electrophoresis can separate proteins in very complex mixtures
31
Mass spectroscopy
A protein is vaporized by a laser beam, yielding charged protein particles
The time they travel for provides a very accurate mass
32
How can an atom with a lone pair of electrons act
A base if it attracts H+
A Hydrogen bond acceptor if it attracts OH or NH groups
A nucleophile if it shares the lone pair to create a new bond (there can be nucleophilic displacement/ substitution and nucleophilic addition)
33
Nucleophile in hydrolysis
H2O
34
Size of proteins
10 000Da to 100 000Da
35
Hydrogen bonds
Electrostatic attractions between a hydrogen bond donor and acceptor (an electronegative atom with available lone pair of electrons such as O or N)
36
Free amino acids
Weak electrolytes due to their amino and carboxylate groups
So in a solution, they will ionize
They also have acid/ base properties so they can pick up and donate protons
37
Amino acid analysis
Separation and detection
Can be qualitative, quantitative or preparative
38
Ninhydrin
Can be used to detect amino acids by turning them purple (or yellow in the case of proline)
39
Amine
NH2
Primary is bonded to one carbon
The secondary is bonded to two carbons
Tertiary is bonded to three carbons
A substituted amine has been bonded by condensation to a carboxylic acid
40
Carboxylic acid
COOH
41
ether
COC
42
ester
Carboxylic acid without the H, not on the end. If it is on the end it is a carboxylate ion
43
Alcohol
OH
Primary alcohol bonded to a carbon which only has one other carbon attached to is
Secondary alcohol is bonded to a carbon which has two other carbons bonded to it
Tertiary alcohol is bonded to carbon with three other carbons attached to it
44
Ketone
The double bond between carbon and oxygen in the middle of a chain
45
Aldehyde
The double bond between carbon and oxygen at the end of a chain
46
nm x 10^-3
micro meters
47
mmx 1^-3
nm
48
DA
1 g/mol (kda = 1000 g/mol)
49
Amino acids that form an alpha helix
Ala, Arg, Gln, Glu, His, Leu, Lys, Met, (Phe does not care)
50
Amino acids that form a beta helix
Trp, Tyr, Val, Ile, Thr, Cys
51
Secondary structure breakers
Gly, Pro, Asn, Asp, Ser
Allow for folding in the tertiary structure
52
Sanger method
Hydrolysis releases amino acid, but also destroys the remainder of the peptide chain. The first amino acid can be tagger the fluorodinitrobenzene (bright yellow)
53
Edmans degration
Allows N terminal amino acid to be removed and reattached using phenylisothiocynate.
Can be done up to 50 times
Coupling requires base
Cyclization requires acid
No hydrolysis (nucleophilic addition)
54
Tyrpsin (selective hydrolysis)
R or K
55
Chymotrypsin (selectiive hydrolysis)
F or T or Y
56
Cyanogen bromide (selective hydrolysis)
Turns Met into Hse (serine with an extra CH2)
57
Overlap method
Using more than one selective hydrolysis chemical, and then putting the puzzle pieces together
58
Easiest/ cheapest way to determine the amino acid sequence
DNA sequencing
59
Primary structure
Amino acid sequence
60
Secondary structure
Regular repetitive patterns, in short sections of the polypeptide chain
Groups connected by a single bond can rotate about the bond axis, this allows the peptide to adopt a variety of shapes.
A peptide bond is rigid, fixed in trans- geometry because it behaves more like a double bond than a single bond
61
Conformations
represent states of a molecule that can be interconverted by bond rotations without breaking covalent bonds.
**More relevant to this course**
62
Configurations
Can only be interchanged by breaking covalent bonds (cis vs trans)
63
Alpha helix
Held together by hydrogen bonds. 3.6 amino acids per turn, minimum of 5 to make it work
64
Tertiary structure
The overall pattern of 3D folding of the whole polypeptide chain., required for their nature (native state)
Non-polar amino acids are enclosed in the core to minimize contact with H2O (hydrophobic effect)
Non- covalent bonding dictates folding pattern and stability (hydrophobic effect and van Der walls effect are the most important)
Covalent bonding links amino acids in a chain in a specific sequence.
Polar interactions important for maintaining structure are usually in the protein interior, rather than on the surface. This includes hydrogen bonds and ion pairs
65
Denaturation
Unfolds proteins, often irreversible, protein function is typically lost on denaturation.
Ways to denature proteins include heat, disruptive solutions, harsh detergents.
66
Tertiary structure options
Alpha helix bundle
Antiparallel beta-sheet (only beta)
Parallel beta-sheet (alpha and beta. Alpha helixes will run in the same direction and beta helixes will run in the same direction). Also, known as an alpha-beta barrel? The beta-sheet forms the central barrel surrounded by the connecting alpha helixes. Helices on both sides of the sheet give the parallel alpha beta-sandwich
67
Disulfide bonds
Form when pairs of CYS- SH groups react with O2, releasing H2O
Makes a very strong covalent bond got help hold protein together. Usually, proteins designed to function outside cells since O2 needed. Eg. blood cells
68
enzymes
Catalysts
Bind to an active site on a specific target molecule (substrate)
Will not change the delta g
69
Reactions with out catalysts
The reaction depends on random events
Molecules must collide in the right orientation, require a threshold energy
70
Arrhenius equation
rate= p Z e ^- (Ea/Rt)
```
z= collision frequency
p= probability factor, the probability that collision leads to reaction, related to the orientation of reactants
```
Ea= activation energy. Mist be put into a reaction at initial steps to break or distort bonds
e^- Ea/Rt= reaction of molecules at temp T (in kelvin) which possess Ea
71
Proximity effect
Enzyme holds substrates together for long enough for the reaction to proceed (increases Z)
72
Orientation effect
Reactive groups are ideally aligned (increases p)
73
Enzymes lowering Ea
Ea can be lowered by finding a better chemical pathway for the reaction, involving reactive groups on the enzyme
74
Types of chemical catalysts
Nucleophilic catalysts
Electrophilic catalysis
General acid catalysis
General base catalysis
75
Stabilizing the transition state
Reactions must pass through a transition state to proceed where shapes change. The enzyme can help by binding the substrate in the ideal shape for the transition state.
Less activation energy is needed if the enzyme active site is complementary to the transition state
76
Nucleophilic catalysis
Speed up the reaction by providing a better nucleophile
77
Electrophilic catalysis
Speed up the reaction by providing a better electrophile
78
General acid catalysis
Catalysis by an amino acid side chain that donates H+ to the reaction
79
General base catalysis
Catalysis by an amino acid side chain that removes H+ from the reaction
80
Christian Anfinsen
Won a Nobel prize for this study. "The primary stricture contains all the information required for the secondary and tertiary structure of a protein."
Rnase was treated with urea (unfolds protein) and 2-mercaptoethanol (reducing agent, breaks disulphide bonds)
When
A) Urea is removed and
B) O2 is added,
The protein can refold into its native state.
** This will fold wrong if the O2 is added first**
81
Hydrolysis of peptide bonds by chymotrypsin
Binds upstream the targetted amino acid
82
How is chymotrypsin so efficient
Uses two simple steps instead of one complex one, as well as a catalytic triad
83
Catalytic triad
Inside activation site
ASP102
HIS57
SER195
84
1st nucleophilic attack
HIS 57 becomes a general base, takes H+ from SER195
SER195 becomes a better nucleophile
ASP102 delocalizes the positive charge on HIS 57
85
Formation of the 1st transition state
Oxyanion hole pulls O- into the transition state
Favours tetrahedral carboxylation configuration
86
Breakdown of first transition state, formation of acyl- enzyme intermediate
NH group of substrate acts as the leaving group
HIS 57 acts as a general acid, donating H+ to to the leaving group
This NH group remains covalently bond (acyl enzyme)
87
2nd Nucleophilic attack
HIS 57 becomes a general base
Water becomes a better nucleophile (attacks enzyme C=O)
88
Formation of the second transition state
Oxyanion hole stabilizes this configuration
89
Breakdown of the second transition state, formation of products
HIS 57 acts as a general acid, donates H+ to SER 195
Breaks the acyl enzyme bond
N- terminal peptide leaves
Catalytic triad is regenerated
90
Enzyme assay
Process of measuring enzyme catalyzed reaction rate
91
Enzyme kinetics
Mathematical analysis of how rate varies with concentration. "Project curve" is studied
92
Beer lambert law
A= e.c.l
```
A= Absorbance
e= constant
l= thickness (1cm)
c= concentration
```
93
Rate of reaction
change in substrate or product / unit time (M or microM)
94
Enzyme activity
Rate x volume
95
Specific activity
enzyme activity/ mass of enzyme
Can be comparing efficiency or purity
96
Molar activity
Specific activity x molar mass of enzyme
97
Turnover number
Equal to molar activity, number of catalytic reaction cycles per molecule of enzyme per second
98
Thio
S replaces O
99
Enzyme inactivator
Irreversible
| The covalent bond between enzyme and inactivator
100
Enzyme inhibitor
Decrease enzyme activity without destroying catalytic activity
Non- covalent binding
More economical for a cell to make and destroy a small inhibitor than a large enzyme
Many drugs are enzyme inhibitors
101
Competitive inhibition
When inhibitor can only bind to unoccupied enzyme [E]
No effect on Vmax, Km is increased
102
Uncompetitive inhibition
The inhibitor can bind to E and ES
Inhibitor site is different from substrate binding site
V'max decreases as I increases Km is unchanged
103
Distance between each turn of helix
5.4 A (1.5A per amino acid as there are 3.6 amino acids per turn)
104
alpha keratin major pattern
5.4
105
Alpha keratin minor pattern
1.5A
106
B keratin major pattern
7.0A
107
B keratin minor pattern
3.5A
