Exam 1 Flashcards
What is the general structure of amino acids?
Amino acids have a chiral α carbon, an amino group, a carboxyl group, and a variable R group.
Glycine: achiral
20 amino acids are encoded by DNA, and each has a different R group which determines its structure and function.
How does pH affect charge?
pH controls the charge of molecules by affecting protonation states.
Lower pH = acidic = fully protonated = favors positive charge
Higher pH = basic = fully deprotonated = favors negative charge.
Carboxyl (-COOH pKa ~2-4):
Protonated (-COOH) at low pH → neutral charge.
Deprotonated (-COO⁻) at high pH, lowest pKa, → negative charge.
Amino (-NH₃⁺ pKa ~9-11):
Protonated (-NH₃⁺) at low pH → positive charge.
Deprotonated (-NH₂) at high pH → neutral charge.
What type of amino acids are used in natural proteins?
Natural proteins use only L-type amino acids. Translation on ribosome.
R-type aa non-ribosome: bacterial cell wall and antibiotics
This is due to the chirality of the α carbon.
What is a zwitterion?
A zwitterion is a molecule that has both positively and negatively charged groups.
Amine: basic; positive charge; gained proton
Carboxyl: acidic; negative charge; lost proton
Amino acids usually exist as zwitterions at physiological pH.
Protein MW
Formula: MW≈(Number of amino acids)×(110 Da)
ie: The molecular weight of chymotrypsin is approx 25 kDa (kilodaltons) convert to Da, 25,000.
The average molecular weight of an amino acid is about 110 Da (Daltons).
25,000 Da / 110 Da per/amino acid≈227 amino acids
This rough estimate ~241 aa
What is the isoelectric point (pI)?
The isoelectric point (pI) is the pH at which a compound has a 0 net charge.
What is the average molecular weight of amino acids?
The average molecular weight of all amino acids is approximately 110 Da.
The weights range from 75 Da (Gly) to 204 Da (Trp).
What are the four levels of protein structure?
The four levels of protein structure are:
* Primary (1°)
* Secondary (2°)
* Tertiary (3°)
* Quaternary (4°)
What is the primary structure of proteins?
The primary structure is the sequence of amino acids linked by peptide bonds. Covalent bonds
What type of bond forms between amino acids during protein synthesis?
Peptide bonds form between amino acids during a condensation (dehydration) reaction.
What stabilizes α-helices in proteins?
α-helices are stabilized by hydrogen bonds between the carbonyl of residue n and the amide protons of residue n+4. Occurs every four residues apart. This alignment creates a stable, right-handed helix with a repeating structure.
What is the difference between parallel and antiparallel β-sheets?
In parallel β-sheets, the chains run in the same direction, while in antiparallel β-sheets, they run in opposite directions.
What is cooperativity in the context of hemoglobin?
Cooperativity refers to the increased affinity of hemoglobin for oxygen as more oxygen molecules bind to it.
What are the key structural features of immunoglobulins?
Immunoglobulins have a Y-shaped structure with two heavy chains and two light chains, forming antigen-binding sites.
2 heavy chains: each 3 constant domains (CH1, CH2, CH3) + 1 variable domain (VH)
2 light chains: each 1 constant domain (CL) + 1 variable domain (VL)
Fab: fragment antigen binding, top part
Fc: binds to Fc receptors on macrophages & signals to get eaten, bottom part
Can be separated by a protease (papain)
Is bivalent = 2 antigen (Ag) binding sites
each binds to an “epitope” on the antigen
How does ELISA work?
ELISA (Enzyme-Linked Immunosorbent Assay) detects the presence of antigens or antibodies using enzyme-linked antibodies.
What is the role of myoglobin?
Myoglobin stores oxygen in tissues, particularly in muscle, where there is a high demand for oxygen.
What is the role of hemoglobin?
Hemoglobin transports oxygen from the lungs to tissues and carries carbon dioxide and protons back to the lungs.
What is the significance of Ramachandran plots?
Ramachandran plots illustrate the allowed dihedral angles (phi and psi) in a polypeptide chain.
What is the typical structure of fibrous proteins?
Fibrous proteins mainly serve structural roles and are often insoluble in water, having high hydrophobic amino acid content.
What characterizes globular proteins?
Globular proteins have unique tertiary structures and are typically soluble in water. 25% empty, 75% filled
How are quaternary structures formed?
Quaternary structures are formed by multiple polypeptide chains (protomers) that oligomerize into a complex. Non-covalent bonds except disulfide.
What is the significance of peptide bond planarity?
The planar nature of peptide bonds restricts the possible conformations of a polypeptide chain.
Fill in the blank: The polar head groups of molecules arrange to maximize their interaction with _______.
water
True or False: The secondary structure of proteins includes α-helices and β-sheets.
True
What is the molecular weight conversion trick for estimating protein mass?
Covert MW in kDa to Da and Divide the MW by 110 Da
The average molecular weight per amino acid is approximately 110 Da.
What types of interactions establish secondary protein structures?
Secondary structures are established by non-covalent interactions, including hydrogen bonds.
What is the equilibrium expression for the reaction involving protein (P) and ligand (L)?
K = [PL] / [P][L]
K represents the equilibrium constant for the formation of the complex.
What is the association constant (k_a) formula?
k_a = k_on / k_off
k_on=k_a
k_off=k_d
It measures how fast the protein and ligand come together.
What does the dissociation constant (k_d) indicate?
k_d = k_off / k_on
It measures how fast the complex PL breaks apart.
What is the relationship between the dissociation equilibrium constant (K_d) and the association equilibrium constant (K_a)?
K_d = 1 / K_a
When [L] = K_d, what fraction of binding sites are occupied?
θ = 0.5 (50% of binding sites are occupied)
What happens when [L] ≫ K_d?
θ ≈ 1 (Almost all sites are occupied)
What happens when [L] ≪ K_d?
θ ≈ 0 (Almost no sites are occupied)
What is the equation for the fraction of binding sites occupied (θ)?
θ = [L] / ([L] + K_d)
What is the significance of the P50 value in relation to myoglobin?
P50 = K_d = [O2] that results in 50% (half saturation) of Mb
What is the role of myoglobin during exercise?
Myoglobin accepts oxygen released from hemoglobin and delivers it to the mitochondria.
What is the pO2 level in lungs/gills where hemoglobin is present?
pO2 high (100 mm Hg, 13.3 kPa)
What is the pO2 level in tissues where myoglobin is present?
pO2 low (30 mm Hg, 4.0 kPa)
What is the heme group and its function in proteins?
The heme group consists of Fe2+ bound within heme, which is buried within the structure of a globin.
What are the two states in which hemoglobin can exist?
- T (tense) state without O2
- R (relaxed) state with O2
What model explains the conformational change of hemoglobin during oxygen binding?
The Monod/Wyman/Changeux (MWC) model
What is the major class of antibodies?
IgG (immunoglobulin G)
What are the two main parts of an IgG molecule?
- Fab (Fragment antigen binding)
- Fc (binds to Fc receptors on macrophages) signal
What is the principle of ELISA?
To exploit antibody specificity for rapid screening and quantitation for antigen in samples.
Which amino acid sequence is most likely to adopt an alpha-helical secondary structure?
- CYYYCHYYCSG
- SILMVKLASEG
- PAAPLKPGAPI
- KASARDVLMEL
- SWLVIFNWVFG
SILMVKLASEG
What contributes favorably to the stability of a protein structure?
- Covalent bonds between cysteine residues - disulfide bonds
- Higher entropy for water molecules when they are not caged around hydrophobic groups
- Ionic bonds between charged side chains
- Hydrogen bonds between main chain atoms
What does not contribute favorably to the stability of a protein structure?
Lower entropy for the polypeptide when it has a buried hydrophobic core
What is the closest pI for the peptide HAPPEN?
N3H+ His Ala Pro Pro Glu Asn COO-
- 4
- 5
- 8
- 11
- 13
5
What is the net charge of the peptide HAPPEN at pH 5?
0
The average of the two flanking pKa’s (4.25 and 6.00) is closest to 5, making it the best answer.
What are the pKa values for the amino acid side chains in the peptide HAPPEN?
- Arg: 12.48
- Asp: 3.65
- Glu: 4.25
- His: 6.00
- Lys: 10.53
These values are crucial for determining the behavior of the peptide in different pH environments.
Fill in the blank: A lower Kd value indicates _______ affinity.
[higher] affinity.
Fill in the blank: A higher Kd value suggests _______ affinity.
[lower] affinity.
What is the relationship between ligand concentration and the amount of ligand bound to a protein?
The amount of ligand bound to each protein is a function of the ligand’s concentration in solution.
Ligand binding increases with concentration, but the rate and pattern depend on cooperativity and affinity K_d
Hemoglobin binds oxygen cooperatively → sigmoidal curve.
Myoglobin binds oxygen non-cooperatively → hyperbolic curve.
At what pH is the average of the two flanking pKa’s (4.25 and 6.00) closest?
5
What does the term ‘Kd’ refer to in the context of protein-ligand binding?
Dissociation constant
True or False: Proteins bind their ligands with the same affinity.
False
What is the pKa value for the amino acid side chain His?
6.00
What is the pKa value for the amino acid side chain Glu?
4.25
Which amino acid has the highest pKa value listed?
Arg (12.48)
What is the significance of pKa values in peptide behavior?
They determine the ionization state of amino acids at different pH levels.
What is the pKa value for the amino acid side chain Lys?
10.53
What is the pKa value for the amino acid side chain Asp?
3.65
Purification of proteins and complexes
What is “Purification from native tissue”?
- Purify complexes from native sources by isolating a protein directly from the tissue or organism rather than expressing it recombinantly in bacteria, yeast, or mammalian cells.
- pro: native complexes present
- con: yields often limiting, difficult to engineer
Centrifugation: Cellular components
start: Lyse/homogenize/break cells then: centrifuge
High speed centrifugation of lysed cells produces two components: the supernatant (cytosol) and the pellet (organelles and plasma membrane)
Which of the following is FALSE about the peptide bond formation reaction?
A) Generates one molecule of ATP
B) Releases a water molecule
C) Is a condensation or dehydration reaction
D) Links the carboxyl group of one amino acid to the amino group of another amino acid
E) Happens in the ribosome
Generates one molecule of ATP
Affinity chromatography
- based on protein’s affinity to ligand covalently attached to solid resin
- highly selective — proteins are very selective with ligand they bind to
- can allow for single step purification
- generality greatly enhanced by the ability to generate fusion proteins of your target with a protein with known affinity
Ion exchange chromatography
- based on protein’s charge at a particular pH
- not so selective, since many proteins can
have the same charge at a given pH — but very general - resin in figure has negative charge = binds
positive proteins (cation exchange chromatography) - one can also use positively-charged resins
to bind negatively-charged proteins (anion exchange chromatography)
Size exclusion chromatography
- based on protein’s MW
- not so selective, since many proteins have similar MW
- porous resin with lots of different sized channels; smaller proteins enter resin particles while larger
proteins cannot = longer path for smaller proteins than larger ones, takes more time to go
through column
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis
- proteins are coated with SDS negative charge
- SDS mostly removes contributions due to
conformation denaturant and inherent charge in
the folded protein (instead, now there is equal charge per MW) - SDS denatures proteins, so it does not give information about native structure, folding, or activity.
- You can infer if disulfide bonds exist by comparing reducing vs. non-reducing SDS-PAGE, but SDS-PAGE alone does not confirm their role in the native protein structure.
- this effectively allows separation by MW
- smaller things move faster through the gel matrix — so at the end, low MW at bottom and high MW at top
- resolution ~1 kDa = ~1000 Da
SDS-PAGE Process
- first, electrophoresis to separate proteins by MW
- Then treat with a stain such as Coomassie blue to visualize
- note calibration markers (“ladder”) at left
- example shown here: recombinant protein expression (not a recombinant process)
- start with a complex mixture, become simpler with purification
Oligopeptide
Few AA (like pentapeptide = 5mer below)
Polypeptide
Many AA (Molecular Weight (MW) < 10,000 Daltons)
Protein
Big polypeptide (Molecular Weight (MW) > 10,000 Daltons)
Why aren’t all the aa are suited for an α-helix
The ideal α-helix formers are Alanine (Ala/A), Leucine (Leu/L), Methionine (Met/M), and Glutamate (Glu/E) because they have side chains that fit well into the helical structure and favor hydrogen bonding patterns.
Conversely, Proline, Glycine, bulky, charged, and strongly polar amino acids tend to destabilize α-helices.
“Breaking residues” of α-helix:
* adjacent turns of Glu(E)/Asp(D) (negative charges repel)
* adjacent turns of Lys(K)/Arg(R) (positive charges repel)
* bulky R-groups (W,Y,F)
* Pro - unable to H-bond and restricted dihedral angles
A bigger number of destabilizing charges = less propensity to
take up the α-helical
conformation
Is this peptide helical?
RDFTKEYP
No, b/c the same-charged residue separated by 4 residues (n,n+4): RxxxK; DxxxE; Y, P
How to analyze:
1. Assess Each Amino Acid’s Helix Propensity
Different amino acids have varying tendencies to stabilize or destabilize α-helices. Below is a quick classification:
Strong Helix Formers:
Alanine (Ala, A) → Most favorable Leucine (Leu, L), Methionine (Met, M), Glutamate (Glu, E), Lysine (Lys, K) → Common in α-helices
Neutral or Moderate Helix Formers:
Aspartate (Asp, D), Arginine (Arg, R), Threonine (Thr, T) → Can be present but not always ideal
Helix Breakers:
Proline (Pro, P) → Major helix breaker Glycine (Gly, G) → Too flexible, usually destabilizing
- Analyze the Given Peptide (RDFTKEYP)
- Prediction: Is It Helical?The peptide has some strong helix formers (K, E), which help α-helix formation.
However, Proline (P) at the end is a known helix breaker, meaning it can terminate or severely disrupt an α-helix.
The presence of Asp (D) and Thr (T) can also destabilize the helix due to electrostatic repulsion or H-bond competition. - Conclusion
❌ This peptide is unlikely to form a stable α-helix because:
Proline at the end disrupts the helix. Aspartate and Threonine can interfere with hydrogen bonding. The mix of bulky and charged residues may prevent a stable helix from forming.
Secondary structure: α-helix
- R-groups protrude outward
- the repeating unit is a single turn of the helix extending ~ 5.4 Å (3.6 AA/turn, 1.5 Å rise per AA = 5.4 Å rise/turn)
- 3.6 AA/turn
- H-bonds between carbonyl of residue n and amide protons of residue n+4
- every peptide bond has potential to participate in H-bonding
- Q: What is the exception? PROLIN : lack of free amide proton
What if you made a mutation at (area X) to (AA Y) — would it have [effect Z]?
Effect on Protein Function (Effect Z)
Loss of function: If the mutation disrupts critical interactions. Gain of function: Rare, but could stabilize an active conformation or enhance binding. Neutral effect: If the mutation is conservative (similar size/charge) or in a flexible loop.
Example Cases
Aspartate (D) → Glutamate (E): Similar charge, minimal effect. Glycine (G) → Proline (P): Could disrupt secondary structure due to rigidity. Cysteine (C) → Serine (S): Might eliminate a disulfide bond, reducing stability. Leucine (L) → Arginine (R): Drastic—changes from hydrophobic to positively charged, likely destabilizing.
Ramachandran plots
Describe the Distribution of Secondary Structure in a Protein
- describes the dihedral angles phi (φ) and psi (ψ) of a polypeptide
- Ramachandran plot is required to test the modeled geometry of a protein structure
Why Does Hemoglobin (Hb) Exhibit Cooperativity?
Hemoglobin is a tetrameric protein (α2β2) that transports oxygen in blood. It must efficiently pick up oxygen in the lungs and release it in tissues. Cooperativity helps achieve this:
T (Tense) State: Low O₂ affinity, stabilizing Hb in deoxygenated form. R (Relaxed) State: High O₂ affinity, forming upon oxygen binding. Oxygen Binding Switches T → R: Each O₂ bound increases affinity at remaining sites, making Hb an efficient oxygen transporter.
How is Cooperativity Measured?
- Hill Coefficient (nH)
Measures the degree of cooperativity.
nH > 1 → Positive cooperativity (e.g., Hb, nH ≈ 2.8).
nH = 1 → No cooperativity (e.g., myoglobin).
nH < 1 → Negative cooperativity.
Determined from the Hill plot, where the slope = nH. - Oxygen Binding Curve
- Sigmoidal (S-shaped) Curve: Characteristic of cooperative binding (Hb).
- Hyperbolic Curve: Seen in non-cooperative binding (e.g., myoglobin).
What is Cooperativity?
Cooperativity occurs when the binding of a ligand to one site on a multi-subunit protein influences the binding of additional ligands at other sites.
Positive Cooperativity: Ligand binding increases affinity at other sites (e.g., hemoglobin and oxygen).
Negative Cooperativity: Ligand binding decreases affinity at other sites (less common).
Non-Cooperative Binding: Binding at one site does not affect other sites (e.g., myoglobin).
The Monod/Wyman/Changeux (MWC) model
- Explains cooperative binding (e.g., hemoglobin’s oxygen affinity).
- Assumes only two states, T and R with all subunits switching together.
- Used in allosteric regulation of enzymes and receptors.
What is the structure of IgG?
IgG consists of two heavy chains and two light chains, with a Fab region for antigen binding and an Fc region for signaling.
What does the Fab region of IgG do?
It binds to antigens.
What is the molecular weight of IgG?
150 kDa.
What is the significance of the Kd value in antibody-antigen binding?
Kd as low as 10^-10 M indicates high affinity and specificity.
Fill in the blank: The sickle cell mutation generates a _______ on the surface of Hb that is exposed when Hb is deoxygenated.
hydrophobic patch
True or False: The binding of IgG to antigen is governed by chemical complementarity.
True
What mutation causes sickle cell anemia?
A mutation replacing Glu 6 with Val on the β subunits of hemoglobin (HbS).
What does the sickle cell mutation create on hemoglobin?
A hydrophobic patch that leads to interactions between different Hb complexes, forming long fibers.
What is CO/CN poisoning?
CO binds to Fe^2+ in hemoglobin with ~200x stronger affinity than O2, blocking O2 transport.
What happens to H+ (proton) produced by the carbonic anhydrase reaction in tissues?
It helps facilitate O_2 release by hemoglobin through the Bohr effect.
What form of CO2 accounts for the majority of its transport in the body?
~67% transported as bicarbonate (HCO3-).
What role does carbonic anhydrase play in CO2 transport?
It catalyzes the conversion of CO2 & H2O to H+ & HCO3- in red blood cells.
How does pH affect oxygen binding in hemoglobin?
Lower pH increases [H+] bound to Hb, resulting in O2 release
What percentage of CO2 transport is carried by hemoglobin?
~25% carried by Hb via reversible reactions with amino groups.
What is the Bohr Effect?
The Bohr Effect describes how lower pH in tissues leads to increased H+ binding to hemoglobin (Hb), reducing its affinity for O2 and facilitating O2 release.
