Proteins, Muscles, Antibodies Flashcards
1
Q
What value describes the conformation of a polypeptide backbone?
A
Torsion angles
2
Q
What are the two torsion angles of a polypeptide backbone?
A
⍦ - C-C bond
∅ - C-N bond
-amide bond has little rotation because it is planar and has some double bond character
3
Q
What is a Romachandran diagram?
A
- indicates allowed conformations for a polypeptide (due to side chains only certain are allowed)
- ⍦ vs ∅ plotted
4
Q
What are the two exceptions to the romachandran diagram?
A
Proline and glycine
5
Q
Describe proline’s torsion angles.
A
- ∅ values are restricted to -60
- restricted to one side of the plot, can only rotate on the side that has the H, not on the side with the R-group
- more restrained motion
6
Q
Describe glycine’s torsion angles.
A
- less steric hinderance, allowed angles are greater
- no R group, just 2 Hs
7
Q
Describe the structure of a typical alpha helix.
(handed, residues, pitch, bonds, side chains)
A
- right handed helix
- 3.6 amino acids/residues per turn
- pitch (distance covered per turn) = 5.4Ă
- carbonyl (C=O) of Nth residue is hydrogen bonded to the N-H of the N+4th residues
- side chains point outward and downward
8
Q
How are beta sheets stabilized?
A
- held together by H-bonds
- H-bonds are between neighboring polypeptide chains
9
Q
What are the two types of beta sheets?
A
parallel and antiparallel
10
Q
Describe parallel beta sheets.
A
- polypeptides run in the same direction (N-C) and (N-C)
- H-bonds between the strands are angled
- the crossover connection between strands is longer and goes out of the plane
11
Q
Describe antiparallel beta sheets.
A
- polypeptides run in opposite directions (N-C) and (C-N)
- H-bonds between the strands are parallel
- the crossover connection between strands is shorter and on the same plane
12
Q
What are the two classes of proteins?
A
- fibrous and globular
13
Q
Describe fibrous proteins.
A
- have repeating second degree structures
- are structural proteins: hair, nails, muscle tendons
- keratin and collagen are examples
14
Q
What is the structure of Keratin?
A
coiled-coil
15
Q
Describe a coiled-coil.
A
- 2 alpha helices that wind around each other
- 7-residue repeat a-b-c-d-e-f-g
- a and d are non-polar and aggregate together due to the hydrophobic effect - minimize exposure to water
16
Q
Describe the higher order structure of keratin.
A
- coiled coil dimers
- form protofilaments - which are connected by disulfide bonds (covalent linkages)
- form protofibrils
- form microfibrils (eg. hair)
17
Q
How are disulfide bonds formed?
A
cysteine residues have thiol groups that form disulfide bonds under oxidizing conditions
18
Q
How does the number of disulfide bonds impact keratin?
A
- more disulfide bonds = harder keratin (nails)
- fewer disulfide bonds = soft keratin (skin)
19
Q
How can disulfide bonds in keratin be modified?
A
they can be reduced and reformed = allows for straightening/curing of hair
20
Q
Describe the structure of collagen.
A
- triple helix
- 3 polypeptide chains wound around together
- 30% Gly, 30% Pro or H-hydroxy Pro (Hyp)
- repeating Gly - Pro - Hyp
- Rope-like twist of 3 collagen peptides where each aa interacts
21
Q
What is Hyp?
A
- H-Hydroxy Proline
- Addition of OH group catalyzed by prolyl hydroxylose
- this enzyme requires a cofactor: ascorbic acid
- vitamin c deficiency causes scurvy which is incorrectly formed/weak collagen
22
Q
Why is Hyp important?
A
Hyp is a hydrogen bond donor that is necessary to maintain the strength of the triple helix interaction in collagen
23
Q
Describe collagen crosslinking.
A
- covalent crosslinking reaction between collagen strands
- Lysine is converted to allylysine (amine group converted to an aldehyde)
- Two allylysines form allylysine aldol
- Allylysine aldol + His forms 5-hydroxylysine
- Final product - 4 amino acids cross-linked together
24
Q
Describe globular proteins.
A
have non-repetitive second degree structures
25
What is the tertiary structure of a protein?
* the folding of second degree structural elements
* positions of each atom in a protein, including side chains
* Where is every atom located in 3D space?
26
What are two methods of determining tertiary structures?
x-ray crystallography and NMR
27
What are the 3 steps in x-ray crystallography?
1. Crystallize the protein
2. Obtain diffraction pattern
3. Map electron density
Different values of resolution, lower Ă allows for better mapping of protein
28
What does NMR do?
identifies through space contacts of different functional groups
29
What are the pros and cons of NMR vs X-ray crystallography?
* NMR: smaller proteins only, dynamic (more information about movement)
* X-ray: any size protein, static picture, contraining protein - minimizing movement, only one conformation displayed
30
Describe the quaternary structure of proteins.
* more than one polypeptide chain
* eg. dimer, trimer
* geometry of association of multiple polypeptides
* non-covalent associations
31
What controls protein stability?
* _hydrophobic effect_ - non-polar groups minimize contact with water and coalesce in the middle
* _electrostatic interactions_ - amino acids with + and - charges form salt bridges (non-covalent)
* _disulfide bonds_ - covalent bonds, 2-cysteines oxidize to form disulfide bonds
* _metal ions_ - eg. Zn finger - alpha helix celates to Zn metal to help hold structure together
32
What is the hydropathy scale?
* (-4.5) - (4.5)
* number rating how hydrophobic each amino acid is
* larger = more hydrophobic
33
What conditions can be used to denature a protein?
* heat - increased KE
* pH changes - interfere with electrostatic interactions
* detergent - nonpolar molecules break up hydrophobic interactions
* chaotropic agents - disrupt hydrogen bonding interactions
34
What are two examples of chaotropic agents?
urea, guanidinium
35
What are intrinsically disordered proteins?
* unfolded
* fold when in contact with their binding partner
36
When proteins fold, they go from _______ to \_\_\_\_\_\_\_
* high energy, high entropy
* low energy, low entropy
37
Describe the process of protein folding.
1. Secondary structural elements and local segments of second degree structure form (Rapid process)
2. Tertiary structure forms, second degree elements collapse to form tertiary structure (Slow process)
38
What chart illustrates protein folding?
* folding funnel
* energy x entropy
* local energy minima are the dips
* the lowest point is the most stable native structure
39
What do chaperone proteins do?
* assist with protein folding when stuck in minima
* eg. heat shock proteins (Hsp70/90)
* many of these are ATPases that catalyze the hydrolysis of a phosphate to allow a reaction that would not otherwise occur happen
40
What proteins are essential to muscle contraction?
actin and myosin
41
Describe the structure of muscles.
* muscles are made up of bundles of muscle fibres
* one bundle is made of a parallel array of myofibrils
* one myofibril is made up of repeating sarcomere units
42
Describe the structure of a sarcomere.
* made of overlapping thick and thin filaments
* repeating sarcomere units make myofibrils
43
What changes as muscles contract?
the overlap between thick and thin filaments changes
44
Describe the structure of a thick filament of a sarcomere.
* made up of myosin - 1 thick filament has 100s of myosins
* myosin has 6 polypeptide chains: 2x heavy, 4x light
* N-term of heavy chain is a myosin head
* C-term of heavy chain is a long coiled coil (a-d)
* coiled coil regions stick together and myosin heads stick off
45
Describe the structure of a thin filament of a sarcomere.
* made up of actin
* G-actin (globular/monomeric)
* F-actin (fibrous/polymer)
* polymer forms a double stranded helix
46
How do thick and thin filaments of sarcomeres interact (general)?
* each myosin head binds to one unit on the actin polymer
* thick filament - myosin head
* thin filament - actin
47
What conversion occurs during the process of muscle contraction?
chemical energy (ATP hydrolysis) is converted to mechanical energy (movement of thick and thin filaments against each other)
48
Describe the process of muscle contraction.
* ATP binds myosin head
* Myosin head is released from actin
* Hydrolysis of ATP
* Cocking of myosin head (changes angle)
* Myosin head binds weakly to new actin
* Releases Pi
* Pi then allows stronger binding to actin
* Power stroke - moves thin filament so myosin can be at correct angle
* ADP released
49
What are the two types of immune response?
1. Cell mediated - T-cells
2. Humoral - B-cells secrete antibodies
50
What creates antibodies?
B-cells
51
Describe the structure of antibodies.
* antibodies have 4 subunits: 2x light chains, 2x heavy chains
* they are glycosylated at CHO regions
* light & heavy chains are linked with disulfide linkages, the two heavy chains are also linked with disulfides
52
What makes up the “ends” of the antibody arms?
* both light and heavy chains have _variable_ and _constant_ regions
* the variable regions make up the ends of the antibody arms
53
Describe the light chain of an antibody. What is it important for?
* the variable domain of a light chain has _hypervariable loops_ responsible for antigen binding
* light chains also have a characteristic _immunoglobulin fold_ - 3-4 antiparallel beta sheets linked by disulfides
54
How does antigen binding occur?
* antigen binds to hyper-variable loops through non-covalent interactions: hydrophobic, ionic, H-bonding
* this binding can be super tight
55
Describe the binding affinity of antibodies.
* Kd (binding affinity) = 10-14 - 10-10 M
* concentration of antigen for 50% occupancy
* lower the value the tighter the binding
56
How is antibody diversity created?
* recombination of VDJC units
* somatic mutations
57
Describe our antibody system and how diversity is acquired.
* immune system has a library of antibodies (\>1018 )
* each chain is encoded by multiple genes
* light - VJC
* heavy - VJDC
* each individual B-cell combines different V,J,D,C to make a unique Ab
58
What happens when a B-cell is exposed to an antigen?
* upon exposure to an antigen B-cell proliferates
* somatic mutation create diversity of antibodies
59
What are the 3 types of monoclonal antibody therapies?
1. antibodies directly block the function of our cell surface receptor
2. antibodies recognize something specific to a cancer cell surface then recruits other immune cells like T-cells or macrophages to destroy the tumor cell
3. antibody drug conjugate - antibody delivers toxic drug to tumor cell
60
How do you generate monoclonal antibodies?
1. Have antigen X expressed on tumor cells
2. Inject mouse with antigen X
3. Harvest lymphocytes - B-cells (can't culture B-cells)
4. Fuse these with mouse myeloma cells (they multiply indefinitely)
5. Hybridomas formed (half myeloma, half-B cells) - purify for only hybridomas using selective media (in the media myeloma cells are not viable and only hybridomas survive)
6. Clones that survive are screened for the antibody
61
What are polyclonal antibodies?
* a bunch of different antibodies that recognize the same antigen
* produced by different B-cell clones, recognize many epitopes of the same antibody
62
What are monoclonal antibodies?
* single primary sequence of a light-chain heavy-chain combination
* generated by identical B cells which are clones from a single parent cell - only recognize the same epitope of an antigen
63
What is an epitope of an antigen?
a small site where an antibody binds
