Abstract
Determination of the appropriate conditions for protein crystallization remains a highly empirical process. Preventing protein aggregation is necessary for the formation of single crystals under aggregation-prone solution conditions. Because many amino acids and amino acid derivatives offer a unique combination of solubility and stabilizing properties, they open new avenues into the field of protein aggregation research. The use of amino acids and amino acid derivatives can potentially influence processes such as heat treatment and refolding reactions. The effect of the addition of several amino acids, such as lysine, and several amino acid derivatives, such as glycine ethyl ester and glycine amide, on the crystallization of equine hemoglobin and bovine pancreatic ribonuclease A has been examined. The addition of these amino acids and amino acid derivatives expanded the range of precipitant concentration in which crystals formed without aggregation. The addition of such additives appears to promote the crystallization of proteins.
Highlights
Protein crystallography is of increasing importance for biological research
The use of Glu, Arg and glycine amide (GlyAmd) as additives was most effective for the expansion of the conditions of the precipitant concentration
When the concentration exceeded the precipitant by more than 37.5%, the hemoglobin solution aggregated in the absence of any additives, whereas single crystals appeared in the presence of Lys, Arg and GlyAmd, even at 40% of PEG 3350
Summary
Protein crystallography is of increasing importance for biological research. The rapid determination of protein structures has been achieved by progress in areas such as genetics, purification techniques, detector technology and synchrotron radiation. Crystallography requires single crystals of high quality; an efficient method of obtaining these crystals has been difficult to determine. This difficulty depends on a huge number of parameters that influence protein crystallization, which include protein concentration, pH, ionic strength, type and concentration of precipitant, and the additives in the crystallization solution (McPherson, 1999). Guanidine and urea are well known aggregation suppressors that minimize the hydrophobic intermolecular interaction between proteins. These denaturants increase the solubility of unfolded protein molecules (Buchner & Rudolph, 1991; Rudolph & Lilie, 1996). They are often used as additives for the initial screening, but can decrease the stability of the native state of proteins
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