Practice Questions Flashcards
If you wanted to determine the isoelectric point of a protein based on its primary sequence, what tool would you use?
ProtParam
https://web.expasy.org/protparam/
According to the isoelectric point of a protein, how do you know when it will carry a net positive charge or a net negative charge?
- Below the pI the protein will have a net positive charge because the acidic groups (e.g., carboxyl groups, glutamic acid residues, etc.) are protonated and neutral, while the basic groups (e.g., amino groups) are protonated and positively charged in this acidic environment with high hydrogen ion concentration.
- Above the pI the protein will have a net negative charge because ionizable groups are deprotonated in basic conditions where hydrogen ions are scarce. Acidic groups lose protons and become negatively charged, while basic groups remain neutral.
The protein will carry a net negative charge at the pI.
You have a solution of the above protein in phosphate buffer, pH 8, with 10 mM DTT, and you want to measure the protein concentration based on UV absorbance at 280 nm.
How would you determine which extinction coefficient to use?
ProtParam
https://web.expasy.org/protparam/
You have a solution of the above protein in phosphate buffer, pH 8, with 10 mM DTT, and you want to measure the protein concentration based on UV absorbance at 280 nm.
According to ProtParam, the extinction coefficient is 55350 M-1 cm-1 at 280 nm.
Which amino acids contribute to this extinction coefficient? Why?
- This value assumes that all cysteine residues are reduced (no disulfide bonds), which aligns with the presence of DTT, which is a reducing agent and would reduce disulfide bonds.
- The extinction coefficient is primarily due to the presence of aromatic amino acids, tryptophan, phenylalanine, and tyrosine because these residues have conjugated π-electron systems that strongly absorb ultraviolet (UV) light.
- These amino acids contain side chains with ring structures with conjugated double bonds (π-electron systems) that can absorb UV light.
- These conjugated systems are capable of undergoing π → π* and n → π* electronic transitions when exposed to UV light.
- Non-aromatic amino acids lack these conjugated systems and do not absorb significantly in the UV region.
What tool would you use to find out the common name of protein and the organism it is from (scientific and common name)?
Protein Blast P
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins
Beta-casein (UniProtID: P02666) and beta-lactoglobulin (UniProtID: P02754) are both important proteins found in bovine milk, yet we know much more about the structure of the latter.
How many PDB entries are there for beta-casein, and how many are there for beta-lactoglobulin?
What features of these proteins can explain this discrepancy?
- This discrepancy arises from differences in their structural properties.
- Beta-lactoglobulin is a globular transport protein with a compact and well-defined tertiary structure.
- For example, it contains seven cysteine residues (ProtParam), allowing the formation of disulfide cross-linkages that stabilize its structure.
- According to UniProt, one such linkage may occur between residues 82 and 176.
- On ProtParam, its instability index is 40.12, which is near the threshold for stability.
- A protein with an instability index below 40 is predicted to be stable, while a value above 40 suggests potential instability. This relative stability facilitates crystallization and makes beta-lactoglobulin more amenable to X-ray crystallography, a technique that relies on forming protein crystals for structural analysis.
- In contrast, beta-casein is an amphiphilic molecule that lacks a stable tertiary structure under physiological conditions.
- Unlike beta-lactoglobulin, it has only a single cysteine residue, preventing the formation of stabilizing disulfide cross-linkages within its polypeptide chain.
- Its instability index on ProtParam is 94.12, indicating a highly unstable protein.
- This inherent flexibility aligns with its primary role in micelle formation and stabilization in milk.
- However, this same flexibility prevents beta-casein from forming the ordered structures required for X-ray crystallography. Additionally, its structural variability may reduce its suitability for alternative structural determination methods such as cryo-EM.
Thus, the fundamental structural and functional differences between these two proteins largely explain why beta-lactoglobulin has been studied more extensively in terms of structure.
Where can you find a protein’s instability index, and how can you interpret it?
A protein with an instability index below 40 is predicted to be stable, while a value above 40 suggests potential instability.
ProtParam
If given UniProtID for two proteins and asked to compare their % identity, how would you do this?
How would you determine if either protein is glycosylated?
How would you determine if the proteins could be linked via disulfide cross-linkages?
- Collect their primary sequences from UniProt
- Use Clustal Omega to compare % identity
- UniProt can specify which residues are glycosylated
- ProtParam can indicate if any cysteine residues are present
i.e., if both proteins had one cysteine residue: the proteins could be cross-linked via a single disulfide bond, with each protein contributing one of the cysteine residues. This would depend on the correct orientation, oxidizing conditions, and proximity to occur.
Briefly explain the basis of UV-VIS spectroscopy and the type of information and data it provides, and how this relates to structure and stability.
- Measures light absorbance at specific wavelengths, primarily by aromatic residues like tryptophan, tyrosine, and phenylalanine
- Provides insights into protein concentration and structural changes. Shifts in absorbance spectra indicate folding, unfolding, or aggregation states, as these processes alter chromophore exposure
- Data: Absorbance values (e.g., unitless optical density) at specific wavelenghts, typically 280 nm for proteins.
Briefly explain the basis of IR spectroscopy and the type of information and data it provides, and how this relates to structure and stability.
- Basis: Detects molecular vibrations of functional groups, especially the amide bonds in proteins.
- Data/Relation to Structure and Stability: Amide I and II bands provide detailed secondary structure information, such as the proportion of alpha-helices and beta-sheets. Changes in these bands highlight protein unfolding, misfolding, or aggregation.
- Type of Data Produced: Wavenumber in cm⁻¹ (e.g., 1650 cm⁻¹ for α-helices, 1630 cm⁻¹ for β-sheets), with peak intensity representing structural content.
Briefly explain the basis of circular dichroism spectroscopy and the type of information and data it provides, and how this relates to structure and stability.
- Basis: Analyzes differential absorption of left- and right-circularly polarized light by chiral protein structures.
- Data/Relation to Structure and Stability: Secondary structure composition and transitions during folding/unfolding or interactions with other molecules. For example, α-helices and β-sheets have distinct CD spectra, which can be monitored to assess structural integrity.
- Type of Data Produced: Molar ellipticity values in deg·cm²·dmol⁻¹, plotted as a function of wavelength (e.g., 190-260 nm).
Briefly explain the basis of differential scanning calorimetry and the type of information and data it provides, and how this relates to structure and stability.
- Basis: Measures heat flow associated with protein unfolding as temperature changes.
- Data/Relation to Structure and Stability: Provides thermodynamic parameters like melting temperature (Tm), enthalpy (ΔH), and Gibbs free energy changes (ΔG). These data elucidate thermal stability, folding pathways, and the energetic favorability of native states.
- Type of Data Produced: Temperature (°C) at unfolding transition (e.g., Tm of 60°C), heat flow in kcal/mol, enthalpy (ΔH) in kcal/mol, and ΔG in kcal/mol.
Briefly explain the basis of x-ray crystallography and the type of information and data it provides, and how this relates to structure and stability.
- Basis: Uses X-ray diffraction from crystallized proteins to determine atomic positions.
- Data/Relation to Structure and Stability: Produces high-resolution 3D structural models, revealing atomic interactions critical for stability. Useful for understanding the impact of mutations or ligand binding on structural integrity.
- Type of Data Produced: Atomic coordinates (x, y, z) in angstroms (Å), resolution in Å (e.g., ≤2.0 Å for high-resolution structures), electron density maps.
Briefly explain the basis of cryo-electron microscopy and the type of information and data it provides, and how this relates to structure and stability.
- Basis: Uses electron beams to capture images of flash-frozen proteins in near-native states.
- Data/Relation to Structure and Stability: Resolves structures of large, dynamic protein assemblies. Tracks conformational changes and interactions influencing stability under physiological conditions.
- Type of Data Produced: 3D density maps in angstroms (Å), resolution values (e.g., 3.0 Å for near-atomic resolution), particle images at different orientations.
Briefly explain the basis of nuclear magnetic resonance spectroscopy and the type of information and data it provides, and how this relates to structure and stability.
- Basis: Analyzes magnetic properties of atomic nuclei in a magnetic field to provide structural information.
- Data/Relation to Structure and Stability: High-resolution 3D structures of small proteins in solution, including dynamic conformations and flexibility, which are critical for understanding stability and misfolding.
- Type of Data Produced: Chemical shifts in parts per million (ppm), coupling constants (Hz), and 3D atomic coordinates (x, y, z) for solution structures.
For each tool, specify a use:
BlastP
CLUSTAL Omega
ProtParam
UniProt
PDB
BlastP: protein name
CLUSTAL Omega; % identity comparison
ProtParam: pI; extinction coefficient; instability index; number of each residue
UniProt: where disulfide linkages occur; where glycosylation occurs; protein function; structure
PDB: database
