protein crystallization

Before proteins enter the crystallized state, they are usually in a dissolved state in solution. In this state, the protein molecules are dispersed throughout the solvent (usually water or a buffer solution) and can move freely but may still maintain their native folded structure. The purpose of protein crystallization is to transform these dispersed protein molecules from solution into an ordered solid-state lattice arrangement to form crystals. This transformation is facilitated by finding the right conditions (such as concentration, pH, temperature, and the presence of certain salts or other molecules) that encourage protein molecules to clump together and form this ordered structure.

1. Before crystallization:  Proteins dissolve in solution without solid particles. Protein molecules are evenly dispersed throughout the entire liquid.
2. During the crystallization process:  As conditions change (such as due to steam diffusion), proteins in the solution become supersaturated. This means that the protein concentration in the solution is higher than the normal concentration in the dissolved state. At this point, protein molecules begin to gather together and arrange in an orderly repeating pattern, forming solid crystals.
3. After crystallization:  The system consists of solid protein crystals suspended in the remaining liquid solution. Crystals are not just solid particles, but also have an internally ordered structure.

The purpose of protein crystallization

Protein crystallization is a key technique in molecular biology, allowing scientists to observe proteins at atomic resolution. This nuanced way of looking has provided insights into all aspects of biology and medicine, including:

• Understand protein function and mechanism:  The structure of proteins often reveals their functions. By understanding the shape and arrangement of amino acids in proteins, researchers can infer how proteins interact with other molecules, how they function, and how they regulate their activity.
• Drug design:  Many drugs exert their effects by binding to proteins and regulating their activity. Understanding the precise structure of proteins can help design new drugs that can bind more effectively, thereby achieving better therapeutic effects. This method is called structure based drug design.
• Learn about diseases:  Gene mutations can cause changes in protein structure, which in turn can alter function. By comparing the structures of normal proteins and mutant proteins, researchers can gain a deeper understanding of the molecular basis of many diseases.
• Biotechnology applications:  Understanding protein structure can help design proteins with new functions that can be used for various biotechnological applications, such as enzyme design in industrial processes.
• Evolutionary Insights:  Comparing the protein structures of different species can provide a deeper understanding of the evolutionary process and functional protection of proteins.

protein crystallization methods

Vapor diffusion

Overview:  Steam diffusion is a widely used protein crystallization method that relies on slow concentration of proteins and precipitants. When water evaporates from protein solution to reservoir solution, the concentration of protein and precipitant will increase. Once the solution reaches supersaturation, crystals will form.

set up:  Both the hanging drop method and the sitting drop method involve placing droplets containing a mixture of protein solution and precipitant near a reservoir containing a higher concentration of precipitant. In the hanging drop method, the droplet is placed on an inverted cover glass, which is sealed onto a reservoir. In the sitting drip method, the droplet is located on a platform in a sealed well above the reservoir.

mechanism:  Over time, the water in the droplets evaporates and diffuses into the reservoir. This evaporation concentrates proteins and precipitates in droplets, promoting crystallization under favorable conditions.

batch crystallization

Overview:  In this method, proteins and precipitants are directly mixed together without interference to the system, allowing crystals to form.

set up:  Mix protein solution with precipitant solution in a container.

mechanism:  When the conditions in the mixture become favorable (usually due to slow evaporation or special interactions between protein molecules and precipitants), protein molecules begin to aggregate and arrange in an orderly manner, forming crystals.

microbatch crystallization

Overview:  This is a variant of 'batch crystallization' designed for very small volumes to prevent evaporation.

set up:  Mix a small amount of protein and precipitant in a well, then cover it with a layer of oil (such as paraffin oil or silicone oil) to prevent evaporation.

mechanism:  The oil layer ensures that the conditions inside the droplets remain stable over time. Without the influence of evaporation, proteins and precipitants can interact under controlled conditions to achieve crystallization under appropriate conditions.

Why is protein crystallization difficult?

1. Proteins are complex molecules with different surfaces, charges, and conformations.
2. Finding the right conditions for the crystallization of a specific protein is like looking for a needle in a haystack.
3. Slight impurities can also inhibit crystallization.
4. Some proteins are inherently flexible or have multiple conformations, making their crystallization challenging.

Advantages of automated protein crystallization processing compared to manual processing:

1. Consistency and repeatability: Automation reduces human error.
2. High-throughput: Automation enables testing of thousands of conditions simultaneously.
3. High efficiency: Automated systems work around the clock to speed up processes.
4. Save resources: Using smaller volumes reduces the amount of precious protein sample required.
5. Data Management: Automation systems often come with integrated software solutions for data storage and analysis.