1
Q
State why all isolated amino acids bear at least one positive and one negative charge
A
- Amino group NH₃+ bears a (+) charge
- carboxyl group COO- bears a (-) charge
- this makes isolated amino acids “zwitterion”, having equal + and - charges = net neutral
only the terminal amino and carboxylate groups in a peptide retain their charge. the others are eliminated by the formation of peptide bonds
2
Q
pKa of the carboxyl and amino ends of amino acids
A
COOH (pKa < 7)
NH₃+ (pKa > 7)
when side chain carries no charge:
- charge at pH 1 = +1 (COOH/NH3+)
- charge at pH 14 = -1 (COO-/NH2)
3
Q
Which amino acid(s) are chiral, which are non chiral?
A
all amino acids except for glycine are chiral - glycine’s side chain is a hydrogen, therefore isn’t attatched to 4 separate groups
4
Q
What is pKa? What is the relationship of pKa to strength of an acid?
A
- a measure of protonation + deprotonation relative to pH: an acids tendancy to ionize
(HA → H+ + A-) - ‘strength’ of a weak acid
- pka ↓ with stronger acids
5
Q
relationship between pH and pka
A
when pH < pka → [HA] > [A-]
when pH > pka → [HA] < [A-]
pka - acid dissociation
6
Q
Why is it important to know the properties of amino acid side chains?
A
the different types of amino acids will behave differently, change the secondary/tertiary structures of the protein and serve differnt functions
- hydrophobic amino acids: lack relative functional groups, have mainly hydrocarbon side chains
- polar amino acids: reactive due to presence of functional groups. polar amino acids have side chains that contain an electronegative atom
- charge amino acids: possess an electric charge due to the presence of ionizable groups in their side chain
7
Q
hydrophobic amino acids & their 3 letter cods
A
- Alanine (Ala)
- Valine (Val)
- Phenylalinine (Phe)
- Tryptophan (Trp)
- Leucine (Leu)
- Isoleucine (Ile)
- Methionine (Met)
- Proline (Pro)
8
Q
Polar amino acids & their 3 letter codes
A
- Serine (Ser)
- Threonine (Thr)
- Tyrosine (Tyr)
- Cysteine (Cys)
- Asparagine (Asn)
- Glutamine (Gln)
- Histidine (His)
- Glycine (Gly)
9
Q
Charged amino acids, their charge at pH 7 & 3 letter codes
A
- Aspartate (-), Asp
- Glutamate (-), Glu
- Lysine (+), Lys
- Arginine (+), Arg
10
Q
Exceptions to abbreviations being first 3 letters of the amino acid
A
- Asparagine - Asn
- Glutamine - Gln
- Isoleucine - Ile
- Tryptophan - Trp
11
Q
Why are peptides described as having a “sense of direction”
A
amino acids in a polypeptide are arranged in a asymmetric, specific manner
the c-terminal end of one residue is always connected to the n-terminal end of another residue → creates a peptide amide bond
- this means that a polypeptide always begins with an N-terminus and ends with a C terminus
12
Q
Peptide nomenclature
A
- dipeptide: 2amino acids joined by a peptide bond
- tripeptide: 3 amino acids joined by a peptide bond
- tetrapeptide: 4 amino acids joined by a peptide bond
etc… - peptides/oligopeptides: < 40 residues
- polypeptide: long chain of amino acids, >40 residues
- protein: large polypeptide (or >1 polypeptide) with a biological function
13
Q
primary structure for a polypeptide
A
determines polypeptide function
- sequence of amino acids
- amino acids joined by peptide bonds: rigid & planar
14
Q
explain why amino acids in a polypeptide are called residues
A
they are called “residues” because during the process of forming a polypeptide or protein, each amino acid contributes to the chain by losing a specific part: a water molecule is released when 2 aa’s join by dehydration synthesis or a condensation reaction.
amount of peptides = 1 more than peptide bonds
15
Q
Explain why peptide bonds are planar and rigid
A
- electrons in peptide bonds are somewhat delocalized (two resonance forms)
- peptide bonds therefore exhibit a partial double bond character, with no rotation about the C-N bond
- functional groups are potential H-bond acceptors/donors
- The polypeptide backbone can still rotate around the N-C⍺ and C⍺-C bonds
16
Q
Define the term “polypeptide backbone”
A
- formed by a repeating structure of peptide bonds that link the c-terminus end of an amino acid to the n-terminus of the next
- includes C⍺ atoms and those involved in the peptide bond
- side chains project out from the backbone
17
Q
Define the four major levels of protein structure
A
- primary: sequence of amino acid residues
- secondary: the spatial arrangement of the polypeptide backboke
- tertiary: the three-dimensional structure of an entire polypeptide, including all its side chains
- quaternary: the spatial arrangement of polypeptide chains in a protein with multiple subunits
primary structure determines the 3D structure
3D structure determines function
18
Q
State how the properties of peptide bonds limit the possible conformations a polypeptide can adopt
A
- rotation of polypeptide backbones is limited, therefore folding conformations are limited
- primary structure aims to minimize steric hinderance within the polypeptide
19
Q
Describe structural features of an alpha-helix
A
except for amino acid residues at either end, all backbone CO and NH groups are hydrogen bonded to one another in the helix:
- carbonyl oxygen of each residue forms an H-bond with the backbone -NH four residues downstream: C1…N5 C2…N6 (residue’s 3-4 apart in the primary structure are close in the secondary structure)
- right handed helical structure
- side chains/R groups face outward
- core of helix: comprises completely of the backbone (van der Waals contact with one another in the center)
groups that interact unfavourably (3-4 residues apart) destabilize the sturcture
20
Q
Describe structural features of parallel and antiparallel beta sheets
A
parallel: neighbouring chains run in the same direction
- N → C (same directionality)
- alternating diagonal H-bonds
antiparallel: neighbouring chains run in opposite directions
- N → C, C → N
- parallel H-bonds
**for both: **
- Each residue forms two hydrogen bonds with a neighboring strand - all hydrogen-bonding requirements are met, except in the first and last strands of the sheet
- every 2nd aa will be found on the same side of the sheet
- side chains are located above and below the plane of the sheet (pleated aspect)
again, steric hinderance is minimized and H-bonding is maximized
21
Q
Distingush between regular and irregular secondary structure
A
regular occurs when every amino acid in a segment of the polypeptide adopts the same geometry (alpha helices and beta sheets; maximize H-bonding, minimize steric hinderance)
irregular - does not mean disorder; just means there is no repeating geometry - necessary to form the compact protein structures
22
Q
State how secondary structures are stabilized
A
- ⍺-helices: H-bonds between backbone CO and NH groups in the same helices (within a continous set of aa)
- β-sheets: H-bonds between backbon CO and NH groups of neighbouring strands (aren’t formed with the consecutive strand)
23
Q
Define tertiary and quaternary levels of protein structure
A
tertiary: arrangment of secondary structures in relation to one another (positions of amino acid sidechains, prosthetic groups)
quaternary: proteins composed of more than 1 polyppetide chain - each polypeptide chain is called a subunit
24
Q
fibrous and globular protein structures
A
- fibrous (elongated) - aq insoluble, form long protein filaments, structural or connective proteins (collagen)
- globular (compact) - fold in on themselves, more varied (mix of ⍺ helix, irregular, β sheet), aq soluble, fold into compact structures ith nonpolar cores and polar surfaces
25
State how the surface and core regions of soluble globular proteins differ
* hydrophobic side chains generally reside on the interior of a globular protein
* hydrophilic side chains are most likely to be found on the surface of a globular protein - can interact with water
* hydrophobic effect: predominant force in folding of soluble globular proteins - the shape of the globular proteins depends on the position of the hydrophobic amino acids in the primary structure
26
Identify amino acids that are most likely to be found at the core of a soluble globular protein
27
Explain why irregular secondary structure is more likely to be found at the surface of a globular protein than alpha helices and beta sheets are
secondary structures likewise typically reside on the interior of globular proteins and loops/irregular structures are on the exterior as their H-bonding is not fully satisfied and can interact with water
28
State how the tertiary and quaternary levels of protein structure are stabilized
**tertiary**
* weak H-bonds b/w closely positioned side chains, as well as backbone groups and side chains (fine tune)
**quaternary:**
* stabilized by same forces as 3° structure (hydrophobic int, H-bonds), typically don't include disulphide bridges
29
naming quaternary structures
named by number and type of subunits
- dimer (2), trimer (3), tetramer (4)
- identical subunits: homo - dimer/trimer..
- non-identical subunits: hetero - dimer/trimer...
30
Describe the terms ion pair (salt bridge), H-bond, and disulphide bridge (disulphide bond) and state their roles in protein tertiary structure
**Ion Pair**: electrostatic interactions between closely-positioned charged groups
- fine tune 2° and 3° structures
- positive and negatively charged groups
- (+) N terminus, Lys, Arg, (His @ high pH)
- (-) C terminus, Asp, Glu (Tyr, Cys @ low pH)
**Disulphide bridge**: covalent bonds b/w closely positioned cysteines
- form stabilizing cross-links for extracellular proteins (or proteins in the lumen)
- disulphide bonds maintain structure in extracellular environments
- in the cytosol, cysteines do not oxidize to cystine (it's a reducing environment)
- reducing agents (DTT) can disrupt disulphide bridges
31
Define the term "prosthetic group" and state why proteins need them
a non-peptide component that is permanently incorporated into a protein, these provide:
- structure (Zn2+ in zinc fingers)
- functional chemical groups (heme in hemoglobin)
32
define domain and motif
**domain**: polypeptide egment that has folded into a single structural unit with a hydrophobic core - proteins may contain more than one domain
"sleeve of a sweater"
**motif**: a short region of polypeptide with a recognizable 3D shape
"cable pattern of sweater"
33
What type of interactions cause protein folding
non-covalent interactions:
- hydrogen bonding
- van der waals
- ion pairs
- disulphide bridges
- hydrophobic interactions
34
Define apoprotein and holoprotein
**apoproteins**: polypeptide without prosthetic group
**holoproteins**: polypeptide with associated prosthetic group
35
How do globular proteins get denatured? (4)
1. heat - H-bonds/hydrophobic int
2. changes in pH - salt bridges/H-bonds
3. salt - salt bridges/ion pairs
4. detergents - hydrophobic int
36
Describe and compare the structures of myoglobin and hemoglobin
**myoglobin:** monomer (no quaternary structure)
* heme prosthetic group exists in the hydrophobic pocket between helicies E&F
* porphyrin ring held in place by hydrophobic interactions AND coordination bond between iron2+ and proximal histidine (aa 93) HisF8
1. ~153 amino acids
2. 8 ⍺-helices identified by letters A-H (A is first helix, H is the last helix)
3. irregular structures
3. heme prosthetic group
**hemoglobin:** heterotetramer (has quaternary structure)
* tetramer with two types of globin - 2 alpha, 2 beta subunits
* has 4 heme prosthetic groups
37
state the physiological functions of myoglobin and hemoglobin
**myoglobin**: facilitates oxygen diffusion through muscle tissues, local reserve of oxygen during intense exercise, stores oxygen in aquatic animals (anaerobically)
**hemoglobin**: in red blood cells - binds O₂ in the lungs and releases it in the tissues (aerobic)
*both bind oxygen reversibly but bind it under different conditions*
38
describe structural feautures of heme
* heme is *circular and planar*: porphyrin ring with a Fe²⁺ ion coordinated between four N atoms
* two propionyl groups at the bottom of the ring (polar and charged) - rest of the molecule are non-polar aliphatic groups
* heme is in a hydrophobic pocket between helices E and F
39
what is a ligand and properties of ligands
* small molecule that binds to another molecule
* the greater the affinity of the ligand for the protein, the more of the ligand-protein complex we will have at any concentration of ligand/protein
40
describe the oxygen binding site in myoglobin
- the oxygen binds to the 6th coordinated position on the heme prosthetic group in myoglobin
- binds at an angle to form interactions with the iron atom
the distal histidine (HisE7) assists with O2 binding by:
1. increasing oxygen binding affinity
2. lowering affinity for other molecules (carbon monoxide)
3. increasing specificity for oxygen
41
T/F myoglobin has quaternary structure and prosthetic group is apart of this
False - myoglobin does not have quaternary sturcture. Prosthetic group is apart of the tertiary structure
42
outline the general molecular mechanism behind cooperative binding
binding of the first ligand to the protein increases affinity for the other binding sites located on the protein by inducing a conformational change.
eg. hemoglobins binding sites are not independant but instead communicate with each other in order to work in a unified fashion
43
Describe the general mechanism of action of an allosteric effector
- allosteric effectors: binding of this compound alters the affinity of other binding sites
- homoallostery: binding of the effector causes more binding of the same compound
- heteroallostery: binding of the compound causes more binding of another compounds
- activator/+ effector: increases binding affinity
- inhibitor/- effector: decreases binding affinity
## Footnote
oxygen is a homoallosteric activator of hemoglobin
44
Describe the molecular mechanism by which oxygen causes hemoglobin to switch states (3)
1. no oxygen is bound = T state
2. O₂ binding to an alpha subunit: iron moves into the plane of the heme, HisF8 moves along with the heme, helix F moves.
3. *SUBUNIT INTERFACE CHANGES* all other subunits change to the R state - increasing the binding affinity of other binding sites
45
function of oxygen binding site
oxygen binding sites are designed precisely to optimize binding specificity and affinity
46
define specificity and affinity
**specificity:** the selectivity of a molecule towards it's target, reflecting how precisely a molecule interacts with it's intended partner or target molecule
**affinity:** how strongly a molecule interacts/binds with it's target, how tightly they bind together
47
What is the Bohr effect?
The decrease in oxygen binding affinity of hemoglobin in response to a decrease in pH/increase in H+ atoms
48
Describe the mechanism by which H+ ions stabilize the T state of hemoglobin
1. lowering pH = protonation of side chains and functional groups (**His + H = His+, NH2 + H = NH3+**)
2. groups associated with BPG binding become protonated (+) - ↑ BPG binding, ↓ O2 binding
* lowering pH leads to increased O2 delivery in muscles and lower affinity of hemoglobin for oxygen
49
The effect of BPG on the oxygen binding behaviour of hemoglobin
- stabilizes the T state of hemoglobin
- acts as a (-) allosteric effector of oxygen binding
- increase [BPG] = decreased O₂ binding
50
Outline the mechanism by which BPG exerts its effect on hemoglobin
1. BPG binds to the central cavity in deoxymyoblobin (T-state)
2. BPG negative charges interact with the positively charged groups on the protein that are directed into the central cavity - **2N terminal residues, 4 His, 2 Lys**
3. BPG causes conformational changes to the oxygen binding sites
overall action of BPG is to decrease the affinity of hemoglobin to O₂
51
Identify four specific roles for histidine residues in hemoglobin function
**The proximal histidine (HisF8):**
1) Binds heme into the heme-binding pocket
2) Prevents oxidation of iron atom
**The distal histidine:**
3) ↑ oxygen binding affinity by assisting in binding and ↓ affinity for other molecules (increasing specificity for O₂)
**His in central cavity:**
4) BPG binding
52
Differentiate between conservative and critical amino acid substitutions
function of a protein is determined entirely by it's structure → amino acid sequence is key
Conservative:
- relatively minor effects on structure/function
Critical:
- change structure and function depending on location
53
How do binding curves differ for the oxygen binding behaviour of fetal and adult hemoglobins
fetal hemoglobin - sigmoidal shift to the left = more O₂ affinity and less O₂ delivery
Adult hemoglobin - sigmoidal shift to the right = less O₂ affinity and more O₂ delivery
54
State how fetal hemoglobin differs structurally from adult hemoglobin
- 2 alpha and 2 gamma subunits
- His143 is sub for serine in fetal gamma subunit
55
Explain how the structural difference between fetal and adult hemoglobin results in their different oxygen binding behaviours
His143 is involved in BPG binding
- no His143 = ↓ BPG binding therefore ↑ O₂ affinity
BIOCH 200
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