Protein Function Flashcards

A set of practice flashcards covering protein structure, fibrous and globular proteins, specific proteins like keratin, collagen, and hemoglobin, and protein analysis techniques.

What is the definition of primary (1°) protein structure?

The linear sequence of covalently bonded amino acids.

What characterizes secondary (2°) protein structure?

The local arrangement of the polypeptide backbone in space, which is highly dependent on hydrogen bonds.

What is tertiary (3°) protein structure?

The overall three-dimensional structure, global fold, and shape of a single polypeptide.

What is quaternary (4°) protein structure?

The structure of proteins composed of more than one subunit or polypeptide chain.

Which protein type is characterized as elongated, narrow, and filamentous?

Fibrous proteins.

Which protein type is described as round and spherical?

Globular proteins.

Contrast the solubility of fibrous and globular proteins in $H_2O$.

Fibrous proteins have low solubility, while globular proteins have high solubility.

Are fibrous or globular proteins more sensitive to changes in heat and pH?

Globular proteins are more sensitive to changes in heat and pH.

Give three examples of fibrous proteins listed in the notes.

Collagen, Keratin, and Elastin.

Give three examples of globular proteins listed in the notes.

Haemoglobin, myoglobin, and immunoglobulins.

What are the two broad classes of keratins based on secondary structure?

$\alpha$-keratin and $\beta$-keratin.

What is the predominant secondary structure of $\alpha$-keratin?

Alpha helical.

What is the predominant secondary structure of $\beta$-keratin?

Beta sheets.

Where is $\alpha$-keratin primarily found in humans?

Hair and finger nails.

Why does $\alpha$-keratin form a coiled-coil structure?

It contains large hydrophobic residues every four amino acids, creating a hydrophobic side on each helix that allows two molecules to interact.

How can the rigidity of keratins be increased?

By crosslinking through disulfide bonds between cysteine-rich polypeptide chains.

What characterizes Type I and Type II $\alpha$-keratin?

Type I is acidic and Type II is basic.

What is the most abundant protein in the human body?

Collagen.

What specific cell type produces collagen?

Fibroblasts.

What is the basic structural unit of collagen?

The triple helix (tropocollagen).

How does the tropocollagen helix differ from a standard alpha helix?

It is a triple-strand left-handed helix with $3.3$ amino acids per turn, whereas an alpha helix is a single polypeptide with $3.6$ amino acids per turn.

Which two amino acids act as secondary structure breakers but are rich in collagen?

Proline and Glycine.

What is the repetitive tripeptide motif found in collagen?

$Gly-X-Y$, where $X$ is Proline and $Y$ is Proline or Hydroxyproline.

What is the purpose of hydroxyproline (Hyp) in collagen?

It participates in hydrogen bonding to aid stability.

What is the role of hydroxylysine in collagen?

It serves as a site for carbohydrate modification that helps in fibril formation.

Which vitamin is required as a cofactor for prolyl hydroxylase in collagen synthesis?

Vitamin-C (Ascorbate).

What are the rubber-like properties and function of Elastin?

It can be stretched several times its normal length and recoil to its original shape.

Where is Elastin typically found in the body?

Lungs, walls of arteries, and elastic ligaments.

Unlike collagen, what does Elastin lack in its structure?

A regular secondary structure.

What unique cross-links are formed in Elastin between strands?

Desmosine cross links.

How is the desmosine ring in Elastin formed?

Via the condensation of three oxidized lysine side chains (allysine) and one regular lysine.

What enzyme is released by neutrophils to degrade elastin?

Elastase.

Which protein inhibits elastase to protect tissue integrity?

$\alpha1$-antitrypsin (AAT).

What is the result of an inherited defect in the AAT gene substituting E342 for K?

The AAT protein polymerizes and cannot be secreted from hepatocytes, leading to elastin degradation and disease.

What are the two forms of actin?

G-actin (globular monomers) and F-actin (filamentous polymer).

What molecule must bind to G-actin to induce the conformational change into F-actin?

$ATP$.

What is the structure of F-actin?

Two helical strands of actin wound around each other.

How does ATP hydrolysis affect F-actin stability?

The release of Phosphate ($P_i$) causes a conformational change that destabilizes actin-actin interactions.

What are the two types of immune responses mentioned?

Innate and adaptive.

An IgG antibody is composed of which chains?

Two identical heavy chains and two identical light chains.

What domains in an antibody confer specificity to antigenic determinants?

Variable (V) domains.

What two fragments are produced by the proteolytic separation of an IgG antibody?

$F_{ab}$ fragments and one $F_{c}$ fragment.

Describe the immunoglobulin fold.

A motif composed of two antiparallel $\beta$ sheets stacked face to face.

How many times does the immunoglobulin fold motif occur in an IgG molecule?

12 times.

A hallmark of rheumatoid arthritis (RA) is an increase in serum IgG containing what?

Incorrect sugar residues.

Where are the complementarity-determining regions (CDR loops) located on an antibody?

At the ends of the antibody variable regions.

What is a prosthetic group in the context of proteins?

A non-polypeptide unit required for the biological function of a protein.

Describe the structure of Heme.

A porphyrin ring with four nitrogens holding an $Fe^{2+}$ ion in the center.

Where is myoglobin primarily found?

Heart and skeletal muscle.

What are the roles of the proximal and distal histidines in myoglobin?

Proximal H binds directly to heme; distal H helps stabilize oxygen binding.

Describe the oxygen dissociation curve for myoglobin.

Hyperbolic.

Describe the oxygen dissociation curve for hemoglobin.

Sigmoidal.

What is the quaternary structure of adult Hemoglobin (Hb)?

A $65\,kD$ heterotetramer consisting of $\alpha_2\beta_2$ subunits (a dimer of dimers).

What are the T and R forms of hemoglobin?

T is the Tense form (low affinity for oxygen); R is the Relaxed form (high affinity for oxygen).

What does the term 'positive homotropic effector' mean in the context of hemoglobin?

Oxygen binding increases the affinity of hemoglobin for more oxygen.

How much greater is the affinity of hemoglobin for the last $O_2$ molecule compared to the first?

$300 \times$ greater.

What is 2,3-bisphosphoglycerate (2,3-BPG)?

A negatively charged metabolite of glycolysis that acts as a negative heterotrophic allosteric effector for hemoglobin.

Where does 2,3-BPG bind on hemoglobin?

In a pocket formed by two beta-globulin chains containing positively charged amino acids.

What is the physiological effect of 2,3-BPG?

It decreases hemoglobin's affinity for $O_2$, facilitating oxygen unloading in tissues.

How does 2,3-BPG expression change at high altitudes?

It is elevated to allow greater unloading of oxygen into tissues.

Where does $CO_2$ bind to hemoglobin?

At the N-terminus of the polypeptide chain.

How does Carbon Monoxide (CO) affect hemoglobin?

It binds tightly to the heme iron and shifts Hb to the R state, preventing oxygen unloading in tissues.

What is the subunit composition of Fetal Hemoglobin ($HbF$)?

Two alpha chains and two gamma chains ($\alpha_2\gamma_2$).

Why is it important that $HbF$ has a higher affinity for oxygen than adult Hb?

To facilitate the transfer of oxygen from the mother's blood to the fetus.

What single amino acid substitution causes Sickle Cell Anemia?

The substitution of Glutamate ($E6$) for Valine ($V$) in the $\beta$ strand.

How does the E6V mutation affect hemoglobin physically?

It causes aggregation of Hb into insoluble fibers that distort red blood cells into rigid, sickle shapes.

What is a proposed gene therapy solution for sickle cell anemia?

Restoring expression of fetal hemoglobin at the expense of adult hemoglobin.

What is the general structure of G protein-coupled receptors (GPCRs)?

Seven transmembrane alpha-helices with flexible N- and C-termini.

What are the three subunits of the heterotrimeric G protein?

$G\alpha$, $G\beta$, and $G\gamma$.

What triggers the activation of the $G\alpha$ subunit?

The binding of an extracellular ligand, which causes GDP to be swapped for GTP.

Describe the function of voltage-gated ion channels.

They are regulated by changes in membrane potential; depolarization causes them to open.

How does the Bradford assay determine protein concentration?

It uses Coomassie brilliant blue dye; upon binding protein, the dye shifts from a brown protonated form to a blue form, changing absorbance from $465\,nm$ to $595\,nm$.

Which technique has been used to solve over 75% of known protein structures?

X-ray crystallography.

What is a major downside of X-ray crystallography?

The crystallization conditions may not be physiologically relevant, and it provides only a single snapshot.

What is a requirement for proteins analyzed via NMR spectroscopy?

They must be uniformly labelled with isotopic atoms, typically $^{13}C$ and $^{15}N$.

What is one advantage of NMR spectroscopy over X-ray crystallography?

Proteins are studied in solution, allowing them to display dynamic conformations.

What does PAGE stand for in protein analysis?

Polyacrylamide gel electrophoresis.

How are proteins separated in Native PAGE?

Separation is based on the protein's size and charge in its native, non-denatured conformation.

What is the purpose of SDS in SDS-PAGE?

It denatures the protein and adds a uniform negative charge, allowing separation based only on size.

How does Isoelectric Focusing (IEF) separate proteins?

Proteins migrate through a pH gradient until they reach their isoelectric point ($pI$), where their net charge is zero.

In Size Exclusion Chromatography, which molecules elute first?

Large proteins elute first because they do not enter the tiny pores of the beads.

How are proteins eluted from an Ion Exchange Chromatography column?

By washing with a salt or a pH gradient buffer.

What is the mechanism of Affinity Chromatography?

The stationary phase contains beads with specific molecules (ligand, antigen, etc.) that bind the protein of interest.

What is the function of the distal histidine in Hemoglobin?

It helps stabilize oxygen binding to the iron.

How many polypeptide chains make up Myoglobin?

One (it is a monomer).

What is the molecular weight of Myoglobin?

$18\,kD$.

What is the molecular weight of the Hemoglobin heterotetramer?

$65\,kD$.

What category of proteins provide motility within the cytoplasm?

Motor proteins.

What category of proteins allow cells to bind to each other and the extracellular matrix?

Cell adhesion molecules.

Which fibrous protein is mechanically durable and found in all higher vertebrates?

Keratin.

What amino acid motif allows the triple strand structure of collagen?

The presence of Glycine at every third position ($Gly-X-Y$).

Which enzyme hydroxylates Proline residues in collagen?

Prolyl hydroxylase.

What is the specific change in absorbance measured in the Bradford assay?

A shift from $465\,nm$ to $595\,nm$.

What disrupts the Bradford assay?

Detergents.

What type of bonds hold the light and heavy chains of IgG together?

Disulfide bridges.

What makes an antibody's antigen binding site 'hypervariable'?

It comprises the complementarity-determining regions of the amino acid sequence.

Which amino acid is oxidized to produce allysine in the formation of desmosine?

Lysine.

In affinity chromatography, how is the protein typically removed from the column?

By outcompeting the stationary phase with the addition of a free ligand or using salt/pH gradients.

Which technique is virtually impossible to use for studying large membrane proteins?

NMR spectroscopy.

What defines the 'T state' of Hemoglobin?

Tense state, where subunits are constrained and have low oxygen affinity.