Question 1

Which of the following statements best explains why circular dichroism (CD) spectroscopy can distinguish between α-helix, β-sheet, and random coil secondary structures in proteins?

Accepted Answer

CD spectroscopy measures the difference in absorption of right- and left-circularly polarized light by chiral molecules, and the peptide bond arrangements in different secondary structures produce characteristic spectra.

Question 2

A protein sample analyzed by dynamic light scattering (DLS) shows a single, narrow peak in hydrodynamic radius. What does this indicate about the sample?

Accepted Answer

The sample is monodisperse, containing primarily one species of particle.

Question 3

Which of the following is a limitation of dynamic light scattering (DLS) when analyzing protein quaternary structure?

Accepted Answer

DLS cannot reliably distinguish between a monomer and a dimer unless their hydrodynamic radii differ by at least two-fold.

Question 4

In a chemical crosslinking experiment using BS3, what is the primary chemical group on the protein that reacts with the crosslinker?

Accepted Answer

Primary amines, such as lysine side chains and the N-terminus.

Question 5

Which of the following best describes the main advantage of cryo-electron microscopy (cryo-EM) for studying large, flexible, glycosylated proteins such as the SARS-CoV-2 spike protein?

Accepted Answer

Cryo-EM does not require crystallization and can visualize large, flexible, and heavily glycosylated proteins at near-atomic resolution.

Question 6

During X-ray crystallography, what is the 'phase problem' and how is it commonly addressed?

Accepted Answer

The phase problem refers to the inability to directly measure the phases of diffracted X-rays; it is commonly addressed by molecular replacement or by using heavy atom derivatives.

Question 7

A protein structure determined by X-ray crystallography has a resolution of 1.2 Å. What does this imply about the quality of the electron density map?

Accepted Answer

Individual atoms are resolved, and the structure is considered atomic resolution.

Question 8

Which of the following is a key difference between NMR and X-ray crystallography for protein structure determination?

Accepted Answer

NMR can provide information on protein dynamics and is performed in solution, while X-ray crystallography provides static structures from crystals.

Question 9

In a COSY NMR experiment, what type of correlations are observed?

Accepted Answer

Through-bond correlations between protons separated by up to three bonds.

Question 10

A NOESY NMR experiment reveals a cross-peak between two protons. What does this indicate?

Accepted Answer

The two protons are close in space, typically within about 5 Å.

Question 11

Why is isotopic labeling (e.g., with $$^{15}\mathrm{N}$$ and $$^{13}\mathrm{C}) $$necessary for NMR studies of proteins larger than a few kDa?

Accepted Answer

The abundant isotopes $$^{14}\mathrm{N}$$ and $$^{12}\mathrm{C}$$ are not suitable for high-resolution NMR due to their nuclear properties.

Question 12

Which of the following best describes the information provided by a $$^1\mathrm{H}$$–$$^{15}\mathrm{N}$$ HSQC spectrum of a protein?

Accepted Answer

Each cross-peak corresponds to an amide N–H pair, serving as a fingerprint for the protein's folded state.

Question 13

Why are intrinsically disordered regions of proteins often invisible in X-ray crystallography but detectable by NMR?

Accepted Answer

Disordered regions do not adopt a single conformation in the crystal, so their electron density averages out, but NMR can detect their presence in solution.

Question 14

Which of the following is a limitation of AlphaFold in protein structure prediction?

Accepted Answer

AlphaFold may have difficulty accurately modeling post-translational modifications and alternative conformational states.

Question 15

A wild-type enzyme and a K→A mutant have identical CD spectra at 25 °C, but the mutant unfolds at a melting temperature 17 °C lower and has no catalytic activity. What can be concluded?

Accepted Answer

The lysine is important for stability and/or function, but CD alone cannot distinguish whether its role is structural or catalytic.

Question 16

If two protons are 2.5 Å apart and give a NOE cross-peak of intensity $$I$$, what would be the expected intensity for two protons 5 Å apart, given that NOE intensity falls off as $$1/r^6$$?

Accepted Answer

$$I/64$$

Question 17

Which of the following best explains why visible light cannot be used to resolve individual atoms in a molecule during crystallography?

Accepted Answer

The wavelength of visible light (400–700 nm) is much larger than atomic distances (~1.5 Å), so it cannot resolve features at the atomic scale.

Question 18

Which combination of techniques would be most appropriate for determining the structure of a large, flexible multi-protein complex at near-atomic resolution?

Accepted Answer

Combining cryo-EM for the overall architecture with high-resolution X-ray crystallography or NMR structures of individual components.

Question 19

Why are membrane proteins generally difficult to crystallize for X-ray crystallography?

Accepted Answer

Their amphipathic nature requires them to be kept soluble in detergents or membrane mimics, which complicates crystal formation.

Structural Biology Techniques in Protein Biochemistry

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