Tertiary Protein Structure Determination by X-Ray Crystallography

Tertiary Protein Structure and X-Ray Crystallography

Introduction to Tertiary Structure

• Lecture focuses on tertiary protein structures.
• Uses X-ray crystallography to determine 3D protein structures.
• Learning outcomes:
  • Protein structure.
  • Crystal derivation and protein crystallization.
  • Data collection: Crystallizing proteins to collect diffraction data for determining crystal structures.
  • Case study of structural biology.
• Professor Bogoja Harris credited for lecture notes.
• Structural biology: Focus on 3D (tertiary) protein structures.
• Protein structure hierarchy:
  • Primary: Amino acid chain.
  • Secondary: Local folding (alpha helices, beta strands).
  • Tertiary: Combination of secondary structures.
  • Quaternary: Arrangement of multiple protein subunits.

Importance of Tertiary Structure

• Proteins are involved in various bodily processes, including infections and diseases through interactions with other proteins.
• Examples:
  • Enzymes breaking down substrates in metabolism.
  • Bacterial virulence factors binding to human surfaces.
• Knowing protein structures helps infer their mechanisms and binding sites.
• Drugs can be developed to bind to these sites, inhibiting protein function, or mutations can be introduced to improve enzyme function.
• Proteins are essential for bodily processes and building blocks.
• Understanding protein structures aids in stopping harmful proteins or improving beneficial ones.

X-Ray Crystallography: A Common Approach

• Using protein structure to identify substrate binding sites.
• Drugs can be developed to block protein function by binding to these sites (lock and key mechanism).
• Many modern drugs are developed using X-ray crystallography.

Process of X-Ray Crystallography

• Crystallize the protein.
• Shoot X-rays through the crystal.
• Obtain a diffraction pattern.
• Determine electron density from the diffraction pattern.
• Build a 3D protein structure based on electron density.

Why Use X-Rays?

• Need to diffract light off the object.
• The object must be smaller than the wavelength of light for efficient diffraction.
• Atomic structures require light with a wavelength appropriate for diffracting atoms.
• Atoms are separated by $0.5$ nanometers or $1.5$ Angstroms ( $1.5 ims 10^{-10}$ meters).
• X-rays with a wavelength of $10^{-10}$ meters are suitable for diffracting small molecules, such as atoms.

Obtaining Protein and Growing Crystals

• Obtain protein from sources like E. coli, yeast, mammal cells, or natural sources.
• Separate the protein of interest from other contaminating proteins using chromatography.
• Purify the protein.
• Grow crystals from the purified protein.
• Take crystals to an X-ray source (synchrotron).
• Determine 3D protein structures from the diffraction pattern.

Requirements for Protein Crystallization

• Need concentrated protein, around 1 to 10 mg/mL.
• Protein needs to be reasonably pure to avoid contamination.

Why Crystals?

• Crystals are ordered arrays of molecules.
• Randomly arranged molecules produce weak diffraction patterns.
• Ordered arrangement in crystals amplifies the diffraction signal due to the additive effect of multiple molecules.

Vapor Diffusion Method

• Most common method for growing crystals.
• Involves a sealed compartment with a reservoir solution containing a precipitant (e.g., polyethylene glycol, salt).
• Protein solution mixed with reservoir solution in a drop.
• The precipitant in the drop equilibrates with the reservoir solution, drawing water out of the drop (vapor diffusion).
• As water leaves the drop, the protein solution becomes saturated, leading to crystal formation or protein aggregation.
• Experiments can be set up in hanging drops or sitting drops.

Automation and Robotics

• Robotics (e.g., Gryphon, Mosquito) can handle small volumes of liquid and automate the crystallization process.
• Robotics simplify the process and reduce the amount of protein solution needed.

Screens for Crystallization

• Screens contain a buffer to maintain constant pH and a precipitant to induce vapor diffusion and crystal formation.
• Types of screens:
  • Sparse matrix screen: Unrelated conditions that commonly produce crystals.
  • Rational or grid screen: Similar conditions, such as peg-based or salt-based.
• Commercial screens screen vapor diffusion experiments to identify initial conditions for crystal formation.

Optimizing Crystal Growth

• Manual screens are performed to optimize crystals.
• Crystals are observed under a microscope to monitor growth and improve size.
• Rock imager: A storage cabinet that rotates screens and takes pictures of crystal growth over time.
• The goal is to obtain larger crystals for more intense diffraction data.

Variables Affecting Crystal Growth

• Precipitating agent (PEG or salt).
• Protein concentration.
• pH.
• Additives.
• Temperature.
• Drop size and ratio of reservoir to protein.

Desirable Properties of Crystals

• Large size for more intense diffraction.
• Reproducibility for collecting sufficient data.
• Reasonably quick growth (1-2 weeks).
• Stability for transport and X-ray exposure.

Freezing Crystals for Data Collection

• X-ray beams can damage crystals, so freezing protects them.
• Freezing can cause ice crystals to form, which interfere with diffraction data.
• Cryoprotectants (e.g., glycerol, polyethylene glycol, high salt) prevent ice crystal formation.
• Crystals are frozen at low temperatures (100K) before collecting X-ray diffraction data.

The Experimental Outline

• Crystals are exposed to an X-ray beam, producing a diffraction pattern.
• The diffraction pattern relates to the molecules within the crystal.
• Electron density is derived from the diffraction pattern.
• An X-ray crystal structure is modeled from the electron density.

Synchrotron Facilities

• Provide access to very high-intensity X-ray beams.
• Electrons circulate in a circular motion within a tunnel, guided by magnets.
• Electrons emit X-rays as they move.
• Workstations around the synchrotron collect X-rays.
• X-rays are directed through protein crystals to obtain diffraction data.