Interfacial Aggregation of Bovine Serum Albumin Related to Crystallization Conditions Studied by Total Internal Reflection Fluorescence

✍ Scribed by Alexander N. Asanov; Lawrence J. Delucas; Philip B. Oldham; W.William Wilson

• Publisher: Elsevier Science
• Year: 1997
• Tongue: English
• Weight: 191 KB
• Volume: 196
• Category: Article
• ISSN: 0021-9797
• DOI: 10.1006/jcis.1997.5182

✦ Synopsis

Since the first crystallographic study of a protein (myogloine adsorption and interfacial aggregation of bovine serum albubin) structure (1), X-ray diffraction has become a powerful min (BSA) from solvent conditions that are known to promote and widely used technique for determination of the three-BSA crystallization. In the absence of a crystallizing agent (therdimensional structure of biological macromolecules. The modynamically good solvent conditions), adsorption from a 0.1 mg/mL BSA solution in 50 mM phosphate buffer, pH 6.2, 25ЊC, knowledge of macromolecular structure is important for drug results in formation of not more than monolayer protein films design, understanding of specific interactions of macromoleon both hydrophilic and hydrophobic surfaces. However, in the cules in biological systems, reproducing biological principresence of ammonium sulfate, which is used as an agent to crysples, and/or modifying them in artificial systems that mimic tallize BSA, adsorption yields a relatively thick multilayer protein structure and biological function of proteins. The structural film. The interfacial aggregation is sensitive to hydrophobicity of data are also necessary for the computer modeling of structhe substrate as well as to the prehistory of adsorption. A hydroture/function relationships, for site-directed mutagenesis philic substrate promotes formation of thicker protein films than and for design of artificial macromolecules. Rapid progress a hydrophobic one. For example, in the presence of 2.13 M ammoin these fields stimulates an increasing need for macromolecnium sulfate, BSA adsorption during Ç10 h yields approximately ular crystals, not only of proteins but also nucleic acids, and a 20-monolayer film on the quartz surface, in contrast to a 6-7 multi-macromolecular assemblies.

monolayer film on the hydrophobic surface. Rates of multilayer

To perform the X-ray or neutron diffraction analysis a adsorption and desorption and the ultimate thickness of the multilayer protein film critically depend on the concentration of relatively large (ú0.2 mm) protein single crystal diffracting crystallizing agent. The rates correlate with the osmotic second at high resolution is required. Although in recent years hardvirial coefficient, B 22 , which has been shown to be a predictor for ware and software for X-ray analysis have been improved, protein crystallization. In the range of B 22 values known as a production of suitable protein crystals often remains the bot-''crystallization slot,'' the multilayer adsorption demonstrates a tleneck in structure determination projects. Most proteins are uniform rate of film growth. Noncrystallizing solvents do not exdifficult to crystallize, and crystal growth procedures are hibit this feature. The TIRF technique can be used to investigate generally developed by a laborious trial-and-error screening molecular mechanisms of crystal growth and growth cessation as approach. Some proteins often readily yield small crystals; well as epitaxial and pseudoepitaxial growth using protein crystals however, it can then take years of trial-and-error experimengrown at or attached to the surface. TIRF provides a sensitive tation before high quality and sufficiently large crystals can and powerful tool to probe in situ and in real time the interactions be grown (2).

of dissolved protein with a growing protein solid phase. ᭧ 1997 At the present time there is general agreement among Academic Press protein crystal growers that to rapidly obtain protein crystals Key words: protein adsorption; protein crystal growth; total internal reflection fluorescence; bovine serum albumin; protein crys-with better morphology using a smaller amount of an expentallization; protein aggregation; protein-protein interactions; cryssive biological material a better understanding of protein tallization agent; thin protein films.

crystal growth mechanisms at the molecular level is necessary (3). Progress in this field is hampered, however, by the complexity of the process. More than 20 parameters have been revealed that may affect protein crystal growth