Midterm 1 Flashcards
1
Q
What is the phi and psi value for beta-sheets?
A
phi = -110 deg; psi ~120 deg
2
Q
Are parallel or antiparallel beta sheets stronger?
A
antiparallel
3
Q
What is the length of one antiparallel beta sheet?
A
7A
4
Q
What is the length of one parallel beta sheet?
A
6.5A
5
Q
Where do prolines occur? and why?
A
beta turn– b/c prolines have a kink and would break the beta sheet
6
Q
How many amino acids are invovled in a beta turn?
A
4 amino acids
7
Q
What are the steps of proteins folding?
A
1) formation of secondary structural elements (a-helix, b-sheet)
2) compaction (hydrophobic effect, H-bond)
3) stabilization via bond formation
8
Q
What is the hydrophobic effect?
A
an aggregation of hydrophobic molecules – H2O LESS ORDERED in solution –> HIGH entropy (S) and LOW enthalpy (H) –> free energy = negative, aggregation spontaneous
9
Q
What is the equation for pI?
A
pI = (pka1+pka2)/2
10
Q
What is primary structure?
A
peptide bond
11
Q
What is a partial double bond?
A
planar
12
Q
What is an omega bond?
A
peptide bond
13
Q
What values can an omega/peptide bond only be?
A
can only be 0 or 180
14
Q
What is a psi angle between?
A
C-C
15
Q
What is a phi angle between?
A
N-C
16
Q
What are the phi and psi values for alpha helices?
A
phi = -40 to -140 psi = -47
17
Q
What stabilizes alpha helix?
A
H-bonding
18
Q
What is a right-handed helix?
A
occurs naturally with L-amino acids
19
Q
What is a left-handed helix?
A
rare, easily formed with D-amino acids
20
Q
What is the coiled coil motif of an alpha-helix?
A
hxxhcxc
21
Q
What breaks an alpha-helix?
A
proline
22
Q
What is secondary structure?
A
structures generated by backbone of protein (alpha helices and beta sheets)
23
Q
What type of bonds are mainly responsible for stability of secondary structures?
A
H-bonds
24
Q
What atoms in peptide are involved in H-bonds of secondary structures?
A
carboxyl oxygen and amino hydrogen
25
How many residues per turn of an alpha helix?
3.6 residues per turn of an alpha helix
26
What is a tertiary structure?
3D fold of peptide/subunit
27
What are 4 forces that can stabilize tertiary structure?
1) H-bonds
2) Van der Waals (induced dipoles)
3) disulfide bonds: cysteins
4) ionic bonds/salt bridges
28
What are 2 proteins that do not fold spontaneously/correctly?
chaperons, ubiquitin
29
What is quaternary structure?
arrangement of multiple peptides (>1 polypeptide/subunit)
30
What is the difference between tertiary and quaternary structure?
tertiary: 3D conformation of folded polypeptide
quaternary: 3D structure of multi-subunit protein (multiple tertiary structures together to form protein complex)
31
Do all proteins have tertiary structure?
all proteins have tertiary structure but not all have quaternary structure
32
What is a domain?
minimal folded unit with a specific function
33
What are folds?
minimal folded unit, usually described by secondary structure; independently stable
34
What are motifs?
small sequence associated with a specific job or action
35
Is it possible for a structure to be a domain and a fold? Is it possible for a structure to be a domain, fold, and motif?
yes-- myoglobin is all 3: folded structure "globin fold" is a motif found in all globins; folds into a single domain, which is its entire 3D structure
36
What are the components of a buffer?
1) buffering species
2) salt
3) reducing agent
4) chelator
5) protease inhibitors
6) detergents
37
What is the purpose of a buffer?
protect protein while separating them from other cell components
38
What are the 3 mechanisms of cell lysis?
1) sheer forces
2) impact forces
3) caviation/decompression
39
What is the sheer forces mechanism (cell lysis)?
unaligned forces tear cells apart
40
What is the impact forces mechanism (cell lysis)?
high energy collisions fracture/shatter cells
41
What is the caviation/decompression mechanism (cell lysis)?
sudden and extreme pressure changes cause implosion/explosion of gas bubbles
42
What tool is used to lyse single-celled organisms with cell walls?
sonication probe, french press/microfluidizer, bead beating
43
What tool is used to lyse heart/potatoes?
homogenizer/blender
44
What tool is used to lyse leaf tissue?
LN2 + Pestle and Mortar
45
What tool is used to lyse single celled organisms with no cell wall?
douncer
46
Why do we perform centrifugation?
to separate cellular components by size/density
47
What is differential centrifugation?
use different rotor speeds to separate components
48
What is density gradient centrifugation?
use variation in solute density to separate components
49
What is the purpose of a buffer exchange?
remove unwanted salts/chemicals from protein solution
50
What are the 3 types of buffer exchanges?
1) desalting column
2) dialysis
3) spin column
51
What are the 2 ways to detect protein concentration from the protein that you isolated?
1) colorimetric
| 2) UV absorption
52
What is colorimetric method of detecting protein concentration?
- dye based - change of color
| - Cu2+ based - redox
53
What is the UV absorption method of detecting protein concentration?
Beer's Law: A = ecL
| use W, Y, F (aromatic amino acids b/c fluorescent)
54
What type of proteins are native gels good for?
large multi-subunit proteins
55
What is the main difference between native gels and SDS page?
SDS PAGE denatures proteins while native gels do not
56
How do native gels separate proteins?
migration dependent on size, shape, and instrinsic (or added) charge
57
How do SDS PAGE separate proteins?
separate polypeptide chains based on length
58
What type of proteins are SDS PAGE good for?
good for simple proteins consisting of 1-8 polypeptide chains
59
What are types of enzyme assays used to identify protein of interest?
1) spectrophotometric
2) fluorescent
3) calorimetric
4) chemiluminescent
5) radiometric
6) enzyme coupled assays
60
What is spectrophotmetric enzyme assay?
substrate or product absorbs a specific wavelength (e.g. NADH)
61
What is fluorescent enzyme assay?
substrate or pdt absorbs light at one wavelength and emits light at another
62
What is calorimetric enzyme assay?
heat change of a reaction is measured
63
What is chemiluminescent enzyme assay?
chemical reaction causes light to be emitted (e.g. luciferase, horseradish peroxidase)
64
What is radiometric enzyme assay?
measures the incorporation of a radioactive isotope into substrate or pdt
65
What are enzyme coupled assays?
initial reaction linked to 2nd reaction involving change in absorbance of pdt (e.g. NADH)
66
What is Kd?
1/Ka
67
What is the relationship between Kd and affinity?
high Kd = low affinity
68
What is the curve if protein has coopertivity?
sigmoidsal curve (Hb)
69
What is the fraction of site occupied equation?
theta = [L}/([L] + Kd)
70
When is theta equation true?
true at ANY point on the curve
71
What happens when O2 is released from Hb (IN TISSUES)?
heme ring becomes puckered
72
What happens in cellular respiration?
CO2 is produced, becomes HCO3- and H+
73
What stabilizes T-state (Bohr effect)?
- increase [H+] (decrease pH)
- increase CO2
- increase BPG
74
How does stabilization of T state occur?
stabilization occurs via several salt bridges
75
What happens to CO2 in lungs?
CO2 is expelled from body
76
What happens to O2 in lungs?
O2 binds and heme ring IS NOT puckered
77
Where does R state predominate?
lungs
78
What is n if coopertivity is present?
slope > 1
79
What does it mean in Hill plot if slope = 1?
- only 1 binding site (coopertivity not possible)
- multiple binding sites, but no coopertivity
- substrate [ ] where coopertivity is not occurring due to stabilization of a high or low affinity state
80
What is the Hill coefficient a measure of?
Hill coefficient = measure of coopertivity
81
What does it mean if nH > 1?
coopertivity is present and # of binding sites must be > nH
82
What is an active site most complementary to?
active site most complementary to transition state
83
What is special about high altitude?
high BPG --> allow O2 delivery
84
What is special about fetal Hb?
- binds poorly to BPG
| - maintina high affinity for O2
85
What is a modulator?
a molecule when binding affects the functioning (affinity) of a protein?
86
What does allosteric mean?
binding to one site affects function of other sites
87
What is O2 in terms of allosteric modulator?
-positive = activator --> increases O2 affinity when binding
88
What does homotropic mean?
molecule is the substrate (O2)
89
What are CO2/H+/BPG in terms of allosteric modulator?
-negative = inhibitor --> decreses O2 affinity
90
What does heterotophic mean?
molecule is not the substrate
91
What are permanent ways amino acids can be modified?
- add SeH ("seleno")
- add -OH ("hydroxy")
- add -COO- ("carboxy")
92
What are reversible ways amino acids can be modified?
- phosphorylation ("phospho-")
- methylation ("methyl-"
- acetylation ("acetyl-")
93
What is a holoenzyme?
protein + cofactor
94
What is an apoenzyme?
protein part only
95
What are 3 types of cofactors?
1) prosthetic group
2) coenzymes
3) metal ions
96
What is a prosthetic group (cofactor)?
organic, tighty bound (heme)
97
What are coenzymes?
organic, transient association (loose)
| -ex) vitamins
98
What are metal ions (coenzymes)?
metal (zinc)
99
What are 3 serine endopeptidases?
1) trypsin
2) chymotrypsin
3) elastase
100
What are the characteristics of trypsin active site?
large, acidic residue
101
Trypsin cleaves after which residues?
Arg, Lys
102
What are the characteristics of chymotrypsin active site?
medium and round
103
Chymotrypsin cleaves after which residues?
Phe, Tyr, Trp, Val, Leu (aromatic and nonpolar)
104
What are the characteristics of elastase active site?
small, nonpolar
105
Elastase cleaves after which residues?
Ala, Gly, Ser
106
What is the active site?
substrate/pdt binding site + catalytic site
107
What is the binding site?
- specific charges and shape
- strips away water
- orients substrates optimally for rxn
108
What is the catalytic site?
- provides cofactors and necessary functional groups
| - puts strain on bonds to destabilize
109
What is the equation for enzyme efficiency?
Kcat/Km
110
What is Vo for the Michaelis-Menten equation?
Vo = (Vmax[S])/(Km + [S])
