Crystallization Procedures

3.20.4.1 Theory

Proteins crystallize when water is continuously removed from a protein solution so that the remaining water becomes less mobile. If the proteins manage to associate in an orderly manner in a crystal adopting a state of lower entropy, the protein-embedding water is released and increases the entropy of the solvent. Crystal growth will continue as long as the net entropy change is positive. Crystal nucleation requires a certain degree of supersaturation, which increases the driving force for splitting the solution into a low and a high entropy region. Nucleation is a highly improbable event because a number of molecules have to associate in a productive manner.47'48 Since only a small number of large crystals are required for an x-ray analysis, a few nuclei are sufficient. Therefore, the supersaturation should be limited to allow long periods for the nuclei to develop. As soon as the first nucleus is formed and starts to grow, it depletes the solution, which, fortunately, tends to inhibit further nucleation.

It seems clear that water removal should be slow and should stop at a supersaturation level at which nuclei can form within days. In general, water removal from the protein is slowed by adding a precipitant so that the water is not actually removed but displaced from the protein to the precipitant.49 Convenient precipitants are salts, in particular ammonium sulfate, or small molecular mass alcohols such as methylpentanediol that have the same vapor pressure as water, or inert polymers such as polyethylene glycol that always remain in a state of high entropy.

3.20.4.2 Conditions

Protein crystallization involves the adjustment of numerous parameters giving rise to a multitude of possible conditions. For membrane proteins, detergent has to be added, which increases the number of important parameters even more.50 Each experiment under any given condition requires a certain mixture, the production of which is tedious. No wonder that the provision of these mixtures has now become a profitable commercial enterprise. They are offered in sets of around 100 conditions. For the crystallization of a normal protein about half a dozen of these screening sets are usually tried. The screens reflect general experience gathered over many years of protein crystallization.51 However, they cannot cover every conceivable condition but instead sample the parameter space rather coarsely. If a crystal is found under a given condition, this condition has to be refined by performing fine-tuned sampling around it.

The applied protein concentrations are rarely below 1 mgmL_ 1 or above 10mgmL_1 The pH ranges between 4 and 10 and is usually, but not always, fixed by a buffer. The most successful precipitants are polyethylene glycols with molecular masses of between 400 and 8000 that are chains of 9-180 oxyethylenes, at concentrations of 2-30%. Common detergents are compounds similar to octylglucoside and dodecylmaltoside as well as compounds similar to octyltetraoxyethylene and dodecyltetraoxyethylene. They are usually used at concentrations slightly above the critical micelle concentration (CMC). Detergents with higher polarity such as lauryldimethylaminoxide (LDAO) or even charged detergents such as dodecylsulfate turned out to be unsuccessful precipitants.50

Ammonium sulfate is a popular salt that allows high ionic strength and fits the water structure as it is located at the corresponding end of the Hofmeister series. It is used at concentrations of 0.5-4.0 M. Other suitable salts are phosphates, chlorides, citrates, malonates, acetates, and formates used in the 0.1-3.0 M range. Conditions with divalent cations such as Ca2 + or with chaotropic cations such as Li+ at the other end of the Hofmeister series are frequent. Many conditions include small organic compounds as precipitates. The most popular ones are methylpentanediol (MPD), iso-propanol, ethanol, butanediol, pentanediol, hexanediol, ethylene glycol, and glycerol. The applied concentration range is 10-60%. The rather nonpolar organic solvents, however, tend to denature proteins.

Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment