BioChem exam 2

What type of biochemical test uses antibodies to quantitatively detect the presence of antiviral antibodies in human blood samples.

The test is the "enzyme-linked immnunosorbent assay", commonly known by its acronym ELISA]

Each of the following reagents or conditions will denature a protein. For each, describe in one or two sentences what the reagent/condition does to destroy native protein structure.

(a) urea [is a chaotropic agent that disrupts H-bonds on the polypeptide backbone] (b) high temperature [High temperature changes the G of folding] (c) detergent [solubilizes nonpolar residues that would otherwise fill the protein core] (d) low pH [alters the state of ionization of acidic and basic groups]

What is typically found in the interior of a water-soluble globular protein?

? [Non-polar amino acids]

Explain the mechanism by which antigens bind to antibodies.

Antigens bind to antibodies through an induced fit mechanism]

Describe how you would determine the Ka (association constant) for a ligand and a protein.

I would: 1) label the ligand radioactively or with a fluorescent group; 2) incubate increasing concentrations of the ligand with the same concentration of the protein; 3) separate and quantify the equilibrium fraction of ligand that binds to the protein (B) from the fraction that remains free (F); 4) use the Scatchard formula to determine Ka]

Describe the concept of "induced fit" in ligand-protein binding.

Native protein structures are flexible i.e. they exist in multiple conformations. Upon binding a lingand, non-covalent interactions between the protein and the ligand stabilize one or a few conformations preferentially. The result is ligand-induced fit]

What fraction of ligand binding sites are occupied () when [ligand] = Kd? Show your work.

One half

Describe the resonance structure of a peptide bond, and explain why there is no rotation around the C—N bond.

[The double bond in the carbonyl group C=O delocalizes to a C=N double bond. As a result, the C-N bond is no longer able to rotate freely]

Explain what is meant by motifs in protein structure.

[A motif is a particular arrangement of elements of secondary structure, mainly -helix and b-sheet within a protein]

How does BPG binding to hemoglobin decrease its affinity for oxygen?

[BPG is an allosteric regulator that stabilizes the low affinity T state, facilitating O2 release from hemoglobin in peripheral tissues.]

Pauling and Corey showed that in small peptides, six atoms associated with the peptide bond all lie in a plane. On a dipeptide of two amino acids in trans linkage (side-chains can be shown as —R), which six atoms are part of the planar structure of the peptide bond?

[C, C(carboxyl), O(carboxyl), N(amino), H(amino), C]

Explain how circular dichroism spectroscopy could be used to measure the denaturation of a protein.

[CD measures the difference in specific absorption of UV light with two different directions of polarization. Highly ordered secondary structures within a protein, such as -helix and bsheet have distinct CD spectra. Denaturation causes the loss of these structures and a change in the CD spectrum of a protein]

Explain why carbon monoxide (CO) is toxic to humans.

[CO "poisons" hemoglobin by binding to it with 400 times greater affinity than O2]

Why is carbon monoxide (CO) toxic to aerobic organisms?

[CO binds to the heme-Fe2+ prosthetic group of hemoglobin with approximately 400 times higher affinity than O2. The resulting carboxyhemoglobin is unable to bind oxygen.]

What protein chains make up the Fab regions of antibodies?

[Each antigen binding fragment Fab is made of a part of a heavy chain and an entire light chain]

Identify and quantify the secondary structures present in this protein:

[Four -helices and a single, two-stranded, antiparallel b-sheet]

How can changes in pH alter the conformation of a protein?

[The pH alters the degree of ionization of acidic and basic groups, possibly disrupting electrostatic bridges within the protein, and its interactions with the solvent]

Explain (succinctly) the theoretical and/or experimental arguments in support of this statement: "The primary sequence of a protein determines its three-dimensional shape and thus its function."

[Given that the 20 amino acids have different propensities to form secondary structures, hydrophobicity etc. different primary sequences should not give rise to identical three-dimensional structures. Loss of function mutations that alter the primary sequence provide one kind of experimental evidence of the same]

Why are glycine and proline often found within a turn?

[Glycine has the smallest sidechain group reducing the possibility of steric clashes within the turn, and proline has the greatest propensity of all 20 common acids to assume a cis peptide bond, which allows it to bend the backbone sharply]

Name four factors (bonds or other forces) that contribute to stabilizing the native structure of a protein, and describe one condition or reagent that interferes with each type of stabilizing force. [

[H-bonds, hydrophobic effect, ionic interactions, disulfide bridges, metal-ion bridges]

Describe the quaternary structure of hemoglobin.

[Hemoglobin comprises 2 pairs of and b subunits arranged that alternate around the protein's center]

Where are the hydrogen bonds typically found between two residues in an alpha helix?

[In an alpha-helix the H-bonds form between amino acids that are 4 spaces apart on the sequence]

Explain briefly why the relative affinity of heme for oxygen and carbon monoxide is changed by the presence of the myoglobin protein.

[In the absence of the myoglobin protein, CO binds heme-Fe2+ 20,000 times better than O2. Within the myoglobin protein, the side chain of His E7 forms a hydrogen bond with the O2 in heme-Fe2+-O2 that stabilizes it. Such a stabilizing effect does not exist when CO is bound because the carbon atom in CO cannot form similar H-bond with His E7]

Describe briefly the two principal models for the cooperative binding of ligands to proteins with multiple binding sites.

[In the concerted model, all subunits change state from R to T, or viceversa, at once and ligand binding stabilizes the high-affinity state (R). In the model, each individual subunit can be in either state. With all possible mixtures of states being allowed there is greater conformational diversity]

What would be the expected effect of a mutation that changes the proximal histidine of a heme-containing protein to an alanine on how the heme group is coordinated in the protein.

[It would only have the four coordination bonds to the protoporphyrin ring]

When a polypeptide is in its native conformation, there are weak interactions between its R groups. However, when it is denatured there are similar interactions between the protein groups and water. What then accounts for the greater stability of the native conformation?

[Mostly the hydrophobic effect that results from greater ordering of water molecules around amino acids in the denatured (unfolded) state, followed by stronger electrostatic bonds (H-bonds and slat bridges) due to the lower dielectric constant of the protein core

Explain why the structure of myoglobin makes it function well as an oxygen-storage protein, whereas the structure of hemoglobin makes it function well as an oxygen- transport protein.

[Myoglobin binds O2 non-cooperatively and with very high affinity. Cooperative oxygen binding by hemoglobin reduces its oxygen affinity faster at low pO2 than if the binding where non-cooperative. This increases the fraction of bound O2 that can be released from hemoglobin in the peripheral tissues]

Comparing hemoglobin and myoglobin, explain which has a stronger binding affinity for O2, and why does hemoglobin bind O2 cooperatively while myoglobin does not.

[Myoglobin has the stronger affinity for oxygen. Hemoglobin binds oxygen cooperatively because it has four binding sites (one per subunit), and the binding induced conformational changes in any of its subunits are communicated to the other subunits through the subunits contacts]

Once a protein has been denatured, how can it be renatured? If renaturation does not occur, what might be the explanation? [

[Not every protein can be renatured. Some proteins can be renatured after being denatured under mild conditions, such as with 4-6 M urea. In these cases, renaturation can be achieved by slowly removing the urea through dialysis. The explanation lies in the difficulty to recreate the correct folding pathway for most proteins]

Explain what prosthetic groups of proteins are, and define a common characteristic the applies to all prosthetic groups.

[Organic molecules that are linked, covalently or through coordination bonds, to proteins. They contain chemical groups that confer special reactivities that proteins cannot achieve with the 20 common amino acids alone]

What are two mechanisms by which "chaperone" proteins assist in the correct folding of polypeptides?

[PDI (protein disulfide isomerase) assisted shuffling of disulfide bridges, and ATP-dependent binding to hydrophobic residues]

Briefly describe the mechanism by which pH affects the binding of oxygen to hemoglobin (i.e. the Bohr Effect).

[Protons are released from carbonic acid produced by carbonic anhydrase in peripheral tissues, where they stabilize the low affinity T state, facilitating O2 release. When CO2 excreted in the lungs, the higher blood pH causes the protons to dissociate from hemoglobin O2, which increases its affinity for oxygen]

For the binding of a ligand to a protein, what is the relationship between the Ka (association constant), the Kd (dissociation constant), and the affinity of the protein for the ligand?

[The association constant Ka is the reciprocal of the dissociation constant Kd. Thus Ka= 1 . Both are measures of the protein's binding affinity for a ligand. A greater 𝐾𝑑 Ka means greater affinity. Equivalently, a smaller Kd indicates greater affinity. ]

Two proteins, X and Y, have similar tertiary structures. The quaternary structures of the proteins are indicated by the diagram below. What differences can be expected in the surfaces of the polypeptides X and Y?

[The contact surfaces are hydrophobic, while the surfaces that remain exposed to the solvent in the quaternary structure are hydrophilic]

Explain how the effects of sickle cell disease demonstrate that hemoblobin undergoes a conformational change upon releasing oxygen.

[The sickle cell hemoglobin variants HbS are stable in solution when oxygenated, but the conformational change associated with oxygen release causes them to aggregate into hollow fibers]

In superhelical proteins, such as collagen, several polypeptide helices are intertwined. What is the function of this superhelical twisting?

[To add tensile strength to the structure by close packing of collagen subunits]

Explain why most multicellular organisms use an iron-containing protein for oxygen binding rather than free Fe2+. Your answer should include an explanation of (a) the role of heme and (b) the role of the protein itself.

[When O2 is sandwiched between two heme-Fe2+ groups or when the Fe2+ is free, Fe2+ is readily oxidized by oxygen to Fe3+ which cannot bind O2 .The surrounding protein allows only a single heme-Fe2+-O2 coordinated structure to form, preventing the oxidation of the Fe2+ ion]

Describe three of the important features of a sheet polypeptide structure.

[chain in extended (zig-zag) conformation, neighboring strands H-bond in parallel or antiparallel configuraitons, R-groups are on alternate sides of the plane defined by neighboring strands]

Describe three of the important features of the -helical polypeptide structure predicted by Pauling and Corey.

[right-handed, a period of 3.6 amino acids per turn, R-groups on the outside face]