WEEK 9: Protein Structure and Function Flashcards
(13 cards)
- What is the role of structural biology in understanding protein function?
Model Answer:
Structural biology provides detailed, often atomic-level, insights into protein structures, revealing how conformation influences function, binding, and dynamics. It helps correlate mutations with disease, identify drug targets, and understand conformational changes during activity. Techniques like X-ray crystallography and cryo-electron microscopy (cryo-EM) are central to these insights.
- Differentiate between membrane-bound and soluble proteins and explain the challenges of studying membrane proteins.
Model Answer:
* Soluble proteins exist in the cytoplasm and are generally easier to express and purify.
* Membrane proteins are embedded in lipid bilayers, requiring detergents or nanodiscs to mimic their native environment. Challenges include instability outside membranes, low expression yields, and difficulty in crystallization. Only ~3% of structures in the Protein Data Bank are membrane proteins due to these issues.
- Describe the difference between passive and active transport mechanisms across membranes.
Model Answer:
* Passive transport (e.g., channels, uniporters) moves substrates down their concentration/electrochemical gradient without energy input.
* Active transport requires energy (e.g., ATP or ion gradients) to move substrates against their gradient. Primary active transport (e.g., Na+/K+ ATPase) uses ATP directly, while secondary active transport (e.g., glutamate transporter) uses pre-established ion gradients.
- Explain the principle of two-electrode voltage clamp electrophysiology and its application.
Model Answer:
This technique involves injecting RNA encoding a membrane protein into Xenopus oocytes. Two electrodes measure the ionic current across the membrane to assess protein function (e.g., ion flow via channels or transporters). It’s ideal for studying large currents and membrane protein function in a controlled system.
- What is the purpose of site-directed mutagenesis in studying protein function?
Model Answer:
Site-directed mutagenesis introduces specific amino acid changes to test their functional significance. This allows researchers to:
* Identify key residues in substrate binding or function.
* Confirm predictions from structural or sequence alignment data.
* Investigate disease-causing mutations.
It’s often followed by functional assays (e.g., transport, binding, electrophysiology).
- How does protein crystallography work and what are its advantages?
Model Answer:
Crystallography uses X-rays to analyze protein crystals. Proteins are arranged in a lattice; X-ray diffraction patterns are captured and back-calculated to build atomic models. It provides high-resolution detail, especially for ligand-binding sites. However, it requires large, well-diffracting crystals, making membrane protein crystallization challenging.
- Compare X-ray crystallography and cryo-electron microscopy (cryo-EM).
Requires crystals
Sample type
Protein size limit
Strength
Limitation
X-ray Crystallography
Yes
Ordered 3D crystals
None
High resolution for any size
Hard to crystallize
- CompareX-ray crystallography and cryo-electron microscopy (cryo-EM).
Requires crystals
Sample type
Protein size limit
Strength
Limitation
Cryo-EM
No
Frozen solution
>100 kDa typically
Excellent for large complexes/membrane proteins
Lower resolution for small proteins
- Why is understanding protein structure important for drug design?
Model Answer:
Protein structures reveal active/binding sites, enabling rational drug design to modulate protein activity. Structural knowledge helps design molecules that fit precisely into a protein’s functional region, increasing specificity and efficacy while minimizing off-target effects.
- What is the importance of nanodiscs in structural biology?
Model Answer:
Nanodiscs provide a lipid bilayer-like environment that stabilizes membrane proteins for study. They are composed of a membrane protein surrounded by a lipid bilayer and held together by scaffold proteins. This setup mimics native conditions, preserving protein conformation during cryo-EM or functional assays.
- Outline the general process of obtaining a protein structure from gene to model.
Model Answer:
1. Gene cloning into an expression vector.
2. Protein expression in an appropriate host (e.g., bacteria, yeast, mammalian cells).
3. Protein purification using chromatography, including detergent solubilization for membrane proteins.
4. Structural analysis via X-ray crystallography or cryo-EM.
5. Model building using electron density maps or image averages.
This process can take weeks to years and involves iterative optimization.
- What is a synchrotron and how is it used in protein crystallography?
Model Answer:
A synchrotron is a facility that accelerates electrons to near-light speeds, producing intense X-rays. These X-rays are directed at protein crystals, and the diffraction patterns are collected on detectors. The patterns are then computationally processed to determine protein structure. Australia’s synchrotron is in Melbourne and is used extensively for crystallographic studies.
- Describe the role of protein sequence alignment in structure-function analysis.
Model Answer:
Sequence alignment compares amino acid sequences across related proteins to identify conserved and variable regions. Conserved residues often indicate structural or functional importance (e.g., active sites, ligand binding). This guides mutagenesis experiments to probe protein function.
