Lec 2: Proteins Flashcards
(102 cards)
1
Q
proteins are essentially
A
macromolecules
2
Q
Macromolecules: 4 1* categories:
A
Proteins
Nucleic Acids
Polysaccharides
Lipids
3
Q
Proteins: 9 Major Classes & Functions:
& based on…
A
- ) Enzymes: selective catalysis
- ) Structural Proteins: support of cell structures
- ) Motility Proteins: movement of cells and cell parts
- ) Regulatory Proteins: regulation of cell functions
- ) Transport Proteins: transport of substances across membranes
- ) Hormonal Proteins: communication between distant parts of an organism
- ) Receptor Proteins: response of cells to chemical stimuli
- ) Defensive Proteins: protection against disease
- ) Storage Proteins: storage and release of amino acids
…based on function
4
Q
All proteins are initially…
which =
A
…made as linear polymers of amino acids
= polypeptide chain
5
Q
Amino acids =
A
= monomers
6
Q
Polypeptide chain =
A
= polymer
7
Q
proteins are encoded by:
A
DNA –(transcription in nucleus)–>
mRNA –(translation at ribosome)–>
polypeptide chain –> (folding) –> protein
8
Q
Approximately __ different amino acids contribute to
A
20
proteins (thousands of different proteins in each cell)
9
Q
(AA’s)
Always use the…
A
…L-form amino acid (l-alanine, etc.)
10
Q
(AA’s)
Some are…
usually…
& 3 examples:
A
…modified
(usually via post-translational modification)
ex 1.) proline → hydroxyproline (collagen, plant cell walls)
ex 2.) lysine → hydroxylysine (collagen)
ex 3.) cysteine → cystine (many proteins, dimer of 2 cysteines via disulfide bridge)
11
Q
(AA’s)
General Features: (4)
A
- ) central a-carbon
- ) amino group
- ) carboxyl group
- ) R-group
12
Q
Most amino acids in proteins are
A
L-amino acids
13
Q
(AA structure)
Because the a-carbon is…
it is a…
this gives rise to…
A
…asymmetric (4 different groups attached to it) in most amino acids,
…chiral carbon
…stereoisomers (D and L)
14
Q
Enantiomer =
A
= one of 2 optical isomers
15
Q
Because amino acids differ only at…
it is what determines…
A
…R-Group,
…determines its specific chemical properties
16
Q
(AA’s)
3 main categories of R-Groups:
The R-groups then determine things like…
A
- ) Nonpolar (R is mostly hydrocarbon)
- ) Polar, Uncharged (R contain sulfhydryl, hydroxyl, or carboxamide)
- ) Polar, Charged (R contains carboxyl or amino group)
…charge state, hydrophobicity, possibility for hydrogen bonding, etc.
17
Q
non polar AA’s Side groups are highly
hydrophilic or hydrophobic?
A
organic (hydrocarbon-based)
= hydroPHOBIC
18
Q
polar, charged AA’s contain side groups that have
hydrophilic or hydrophobic?
A
…carboxyl, amino groups or imidazole ring, which are either negatively (acidic) or positively (basic) charged.
= hydroPHILIC
19
Q
polar, uncharged AA’s contain side groups that have…
capable of…
hydrophilic or hydrophobic?
A
…Hydroxyl, sulfhydryl, and amino carbonyls (carboxamide)
…hydrogen bonding
= hydroPHILIC
20
Q
Proteins are formed by
A
successive linkages of amino acids into a polypeptide chain (via the peptide bond)
21
Q
The _______ bond links two successive amino acids via …
A
peptide
…the carboxyl group of one amino acid binding to the amino group of the other amino acid
22
Q
Peptide Bond forms via
A
Condensation Reaction
23
Q
(Peptide Bond Formation)
catalyzed by
A
enzyme peptidyl transferase
24
Q
(Peptide Bond Formation)
occurs at…
during…
A
…at the ribosome
…during translation
25
(Peptide Bond Formation)
| Initially forms a
dipeptide → tripeptide… → polypeptide chain
26
(Peptide Bond Formation)
| Protein =
= final folded version of the polypeptide
27
(Peptide Bond Formation)
| Always starts at...
...the N-terminus proceeds to C-terminus
28
(Peptide Bond Formation)
| cost =
= 4 ATP (or GTP) per peptide bond formed during translation at ribosome
29
Monomeric proteins =
Multimeric proteins =
Homomeric =
Heteromeric =
= proteins composed of a single polypeptide chain
= proteins composed of multiple polypeptide chains
= all subunits are the same polypeptide (ex: LDH-1 & LDH-5)
= different polypeptide chains assemble together (ex: Myosin, hemoglobin, LDH-2, LDH-3, LDH-4)
30
Dimer, trimer, tetramer represents
of subunits
31
What causes the polypeptide to fold in a certain way?
Due to the R-group interactions:
1. ) with other R-groups
2. ) or with surrounding cellular components
32
Polypeptide Folding Possible R-Group Interactions: (5)
1. ) Disulfide bonds (bridges)
2. ) Hydrogen Bonds
3. ) Ionic Bonds
4. ) van der Waals Interactions (forces)
5. ) Hydrophobic Interactions
33
Disulfide bridge =
| what type of bond is this?
= The sulfhydryl (aka thiol) side groups of 2 cysteine combine to form a cystine
= (covalent bond between the two sulfur atoms)
34
Intramolecular disulfide bridge forms a
cross-link
35
Disulfide bridges help to...
...stabilize overall protein folding
36
Hydrogen Bonds involve: (2)
1. ) uncharged, polar interactions
2. ) R–groups with:
- OH
- SH
- C=O
- C-NH2
37
Ionic Bonds Involve...
| making...
...amino acids with charged R-Groups
| ...Opposite charges attract, like charges repel
38
van der Waals Interactions =
= Transient interactions due to transient (+) and (-) charged regions in nonpolar molecules.
39
van der Waals Interactions strength
Very weak and transient electrical based forces between nonpolar molecules.
40
(Hydrophobic Interactions)
Hydrophobic R-groups will tend to...
In contrast, hydrophilic R-groups (charged or polar) will tend to...
...“push” away from charged and strongly polar R-groups and polar molecules (including water)
...be attracted to water and charged R-groups of the opposite charge.
41
2* Protein Structure: (2)
1. ) a-helix
| 2. ) B-sheet
42
a-helix Polypeptide chain forms
a spiral
43
(a-helix)
| R-Groups generally point to...
the outside of the spiral
44
(a-helix)
| Approximately...
four (~3.6) amino acids per complete ‘turn’ of the spiral
45
c
| Small distance between
every 4th peptide bond
46
(a-helix)
| ________ bonding between...
Hydrogen
| ...every 4th peptide bond –strengthens the overall structure (coil)
47
(a-helix)
"MALEK" =
& all have...
= Methionine, alanine, leucine, glutamate, and lysine
| ...especially high helix-forming propensities
48
B-(pleated) sheet =
Extended sheet like conformation with successive peaks and troughs “pleats”
(Like the pleats in a curtain or skirt)
49
(B-sheet)
| involves
two sections of polypeptide positioned side by side
50
(B-sheet)
| ________ bonding between...
Hydrogen
| ...the peptide bonds of the side by side regions helps to stabilize the configuration (not fold)
51
A single section of beta sheet =
= a beta strand
52
2* Structural "Motifs" aka
| examples:
"Combinations"
1. ) B-a-B (parallel)
2. ) Hairpin Loop (anti-parallel)
3. ) Helix-turn-helix
53
Arrow of b-sheet points to
the C-terminus
54
Motifs based on =
| Domains based on =
= structure
| = function
55
3* Structure =
= Overall complex 3 dimensional folding pattern
56
3* Structures are not as well
understood compared to 2* structures (which can be predicted based on AA sequence)
57
(3* Structure)
| Native conformation =
= most stable pattern of folding (under in vivo conditions)
58
2 Main types of 3* structure:
some proteins have...
1. ) Globular proteins (amorphous) [Many enzymes]
2. ) Filamentous (rod-like) [Collagen, elastin]
...both regions
ex: Myosin heavy chain (has a globular head & filamentous tail)
59
4* Structure =
which leads to...
example:
```
= Subunit Interactions
...multimeric proteins
ex: (Myosin)
- 2 Myosin heavy chains
- 4 Myosin light chains
```
60
Protein Domains (sites) result from
3* and 4* structure
61
```
Protein Domains (sites) involve...
& have...
```
...portions of the overall protein
| ...a specific function
62
Many types of domains (examples) (5)
```
DNA binding domain
Substrate binding domain
Regulatory domains
Ligand binding domains
Catalytic domains
```
63
Insulin =
= A protein hormone that is synthesized by the Beta-cells in the Islets of Langerhans in the Pancreas
64
```
(Insulin)
secreted in...
acts as...
without it...
if it becomes ineffective...
```
...the blood
...a chemical signal to cause other cells to take up glucose.
...people suffer diabetes
...leads to insulin resistance
65
(Calcineurin)
| is a...
...Heterodimeric protein
A-subunit = Catalytic subunit
B-subunit = Ca2+ binding subunit
66
(Calcineurin)
| A-subunit Domains:
Catalytic domain
B-subunit binding domain
Calmodulin binding domain
Autoinhibitory domain
67
(Calcineurin)
| B-subunit Domains:
Four EF hand domains
| EF hand dom fun = bind Ca2+
68
Beyond Quaternary Structure, proteins can have...
| Example =
...even higher level multi-protein complexes
| = Thick Filament in skeletal muscle
69
Most (all?) proteins...
...bind to (or at least interact with) other molecules (at least transiently)
70
What ever proteins bind to is called a
| & type of bond?
ligand
| & NON-COVALENT BONDS
71
types of ligands: (6)
1. ) Other proteins (structural, receptors)
2. ) Substrates (enzymes)
3. ) Membranes or membrane components (integral membrane proteins)
4. ) Ions (ionic pumps, Ca2+ activated proteins, etc)
5. ) Infectious agents (antibodies)
6. ) DNA (transcription factors)
etc. ..
72
The portion of a protein that binds something is usually called a
binding domain (or binding site)
73
The ________ of the binding domain for the ligand is dependent on...
affinity
| ...the amino acid composition of that region
74
Binding strength is measured as
the Kd (dissociation constant)
75
(Kd)
The formation of a ligand-protein complex (C) can be described by...
For this reaction, Kd =
... a 2-state process (P = Protein, L = Ligand):
C ↔ P + L
Kd = [P] x [L] / [C]
76
(Kd)
If the reaction tends to favor the bound state (C), then...
If the reaction tends to favor the unbound (dissociated) state, then...
...the Kd will be small = Strong binding
| ...Kd will be large = Weak binding
77
(Kd)
High Affinity =
Low Affinity =
*Note: Do not confuse Kd with
= Low Kd
= High Kd
*the equilibrium constant, Keq
78
Kd is important for understanding...
Kd has units of...
...the functional properties of several types of proteins, including:
1. ) Receptor proteins
2. ) Enzymes
3. ) Transporters
...M (Molarity)
79
(Kd Example)
Protein P and drug D1 have a Kd = 0.1
Protein P and drug D2 have a Kd = 0.01
*Which drug has a stronger interaction with protein P?
drug D2
| since it has a lower Kd = higher affinity
80
At neutral pH the carboxyl and amino groups are...
...charged = Zwitterion (hybrid ion with 1 + and 1 - charge)
81
Zwitterion =
= molecule with formal positive and negative charges on 2 different atoms and a net charge = 0
82
@ pH 1 (acidic)
Low pH
High [H+]
Positive (+) charge
83
@ pH 7 (neutral)
Neutral pH
Neutral [H+]
Net neutral charge
Zwitterion state
84
@ pH 11 (basic)
High pH
Low [H+]
Negative (-) charge
85
The electrical charge on the amino acid is dependent on
the pH
86
However, in a polypeptide most of the carboxyl and amino groups are used for...
What can be charged?
Several R-groups can also be...
```
...the peptide bonds and cannot be ionized
The carboxy (C-) and amino (N-) termini can be charged
...charged (we saw these previously), but the pH will influence their charges in the same way.
```
87
(Aspartate & Glutamate (acidic))
| @ low pH:
high [H+]
R-group carboxyl saturated with H
R-group uncharged
88
(Aspartate & Glutamate (acidic))
| @ neutral or high pH:
low [H+]
R-group carboxyl unsaturated with H
R-group (-) charged
89
(Lysine & Arginine (basic))
| @ neutral or low pH:
high [H+]
R-group amino(s) saturated with H
R-group (+) charged
90
(Lysine & Arginine (basic))
| @ high pH:
low [H+]
R-group amino(s) unsaturated with H
R-group uncharged
91
(Histidine (imidazole ring))
- The charged state of the imidazole ring has a pKa =
- Which means that...
- Therefore a small change in...
- Also, because the pKa is...
- Protein charge depends on...
- Mainly due to...
= 6.8
...at pH 6.8 (near neutral) 1/2 of the histidines will be (+) and 1/2 neutral
...pH (in this range) will have a large influence on the charge of the imidazole ring
...near neutral, imidazole (histidines in proteins) can help “buffer” small changes in [H+]
...pH!
...Asp, Glu, Lys, Arg, (His)
92
Each protein will have: (3)
However, at some pH, the positive and negative charges will...
The pH at which the protein is neutral is called...
1. ) One Amino Terminus
2. ) One Carboxyl terminus
3. ) Variable number of charged amino acids
...cancel each other out and the protein will have no net charge
...the Isoelectric Point (pI)
93
Different proteins have different
isoelectric points
94
You can use pI to...
| & 2 methods:
...isolate proteins
1. ) Electrophoretically (isoelectric focusing)
2. ) Solubility
95
(pI)
| The solubility of a protein in an aqueous environment will be lowest when...
...it has no net charge
96
Isoelectric Focusing allows for
the separation of proteins based on their inherent pI’s
97
*Note: At pH of 9 (high pH, low [H+]) carboxyl groups will be... Thus, the proteins will tend to...
...negative and amino groups uncharged
| ...be negatively charged and move to the positive pole
98
You can also separate proteins based on their...
| by using...
...size (molecular mass)
| ...SDS-PAGE
99
Proteomics =
| “proteome” is derived from...
= refers to all the proteins produced by an organism (or cell or tissue or even cell component)
...proteins expressed by a genome
100
(Proteomics)
| The identification of all the proteins in a specific proteome has largely depended on...
...knowing the pI and MW of each of the constituent proteins
101
```
Traditional method =
Separate proteins based on...
Then separate them in a second dimension based on...
Allows for...
And allows for...
```
= 2D gel electrophoresis
...pI (isoelectric focusing)
...their molecular mass
...identification of a tremendous number of specific proteins in any one sample
...the identification of changes in any one component of the proteome under any experimental condition
102
Other methods for studying proteins: (7)
1. ) Native gel electrophoresis
2. ) Western blotting
3. ) Immunohistochemistry
4. ) ELISA
5. ) Capillary electrophoresis
6. ) Mass Spectrometry
7. ) Protein Arrays
