Question

Which structural biology method (CD, x-ray crystallography, NMR, or cryo-EM) is best suited to each task? a. Obtaining an ultra-high resolution \((<1.5 \AA)\) structure of a drug bound to its protein target b. Obtaining a low-to-medium resolution (5-10 \AA) reconstruction of the \(11 \mathrm{MDa}(11,000,000 \mathrm{Da})\) bacterial flagellar motor c. Identifying the protonation state and \(\mathrm{p} K_{\mathrm{a}}\). of a His side chain in an enzyme active site d. Determining whether a protein is intrinsically disordered or contains secondary structure elements

Short answer

a. X-ray crystallography; b. Cryo-EM; c. NMR spectroscopy; d. CD spectroscopy.

Step-by-step solution

Determine Ultra-High Resolution Method

For obtaining an ultra-high resolution structure (<1.5 Å) of a drug bound to its protein target, X-ray crystallography is the most suitable method. This technique allows for atomic-level resolution and is commonly used for studying well-ordered crystalline proteins.

Select Method for Low-to-Medium Resolution

To reconstruct a complex such as the 11 MDa bacterial flagellar motor at low-to-medium resolution (5-10 Å), cryo-electron microscopy (cryo-EM) is appropriate. Cryo-EM is well-suited for analyzing large macromolecular complexes and provides detailed reconstructions without the need for crystallization.

Identify Method for Protonation State

For identifying the protonation state and \(\mathrm{p} K_{\mathrm{a}}\) of a histidine residue in the active site of an enzyme, NMR spectroscopy is ideal. NMR can provide detailed information about the electronic environment and protonation states of individual atoms within a protein.

Discover Protein Structure Characteristics

To determine whether a protein is intrinsically disordered or contains secondary structure elements, circular dichroism (CD) spectroscopy is appropriate. CD can distinguish between different secondary structures like alpha-helices and beta-sheets, as well as detect disorder.

Key concepts

X-ray crystallography

X-ray crystallography is a powerful tool used extensively in structural biology to determine the three-dimensional structure of crystalline molecules. It involves exposing a crystalline sample to X-ray beams, which diffract when they hit the atoms in the structure. The resulting diffraction pattern is analyzed to deduce the arrangement of atoms.

This method is ideal for obtaining ultra-high resolution structures, especially when studying proteins or drugs bound to their target proteins. In fact, X-ray crystallography can provide resolution finer than 1.5 Å, revealing the positions of individual atoms with high precision.

• It is highly suitable for well-ordered proteins in crystalline form.
• It requires the sample to be crystallized, which can sometimes be challenging for flexible and large complexes.

Despite its tremendous power, the requirement for crystallization is a significant limitation, making some protein complexes unsuitable for study using this method.

cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) has revolutionized the way researchers view large protein complexes and macromolecular assemblies. Unlike X-ray crystallography, cryo-EM does not require crystallization of the sample. Instead, the sample is rapidly frozen to preserve its natural state.

This makes cryo-EM particularly suitable for studying large biological structures at low-to-medium resolution, such as the complex bacterial flagellar motor. It can capture structures around 5-10 Å resolution, which provides a good level of detail for understanding large-scale molecular architecture.

• It allows for visualization of samples in a close-to-native state.
• It is ideal for large molecular structures that are challenging to crystallize.

Cryo-EM provides a snapshot of biological molecules in action and is steadily overcoming the limitations of resolution, bringing finer details into focus.

NMR spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is a versatile tool in the structural biologist's toolkit, used to study proteins in solution. It relies on the magnetic properties of certain nuclei within the molecules.

NMR is particularly useful for identifying the protonation states of residues within a protein, making it ideal for determining the pK_a values of amino acids like histidine within enzyme active sites. Through the analysis of small chemical shifts, NMR provides insights into the electronic environment and dynamics of proteins.

• Suitable for studying proteins that are difficult to crystallize.
• Provides detailed information on protein dynamics and electronic environments.

Despite its advantages, NMR typically requires larger sample amounts and may not be feasible for very large proteins or complexes.

circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy is a rapid and straightforward technique to assess the secondary structure of proteins. It measures the differential absorption of left and right circularly polarized light by chiral molecules.

CD spectroscopy is particularly adept at distinguishing between various secondary structures, like alpha-helices and beta-sheets, making it useful for analyzing protein folding and conformation. It can also determine if a protein is intrinsically disordered versus having a defined secondary structure.

• Quick and simple method to assess protein folding.
• Effective in detecting presence of secondary structure elements like helices and sheets.

While CD provides significant information about secondary structures, it doesn't detail high-resolution atomic positions and must often be used alongside other techniques for comprehensive protein characterization.