Abstract
Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality
Highlights
X-ray crystallography has been the most powerful and robust approach to determining atomic protein structures, which provides incisive insights into the three-dimensional spatial arrangements of protein structures and allows for an in-depth exploration and understanding of the structure-related protein functions [1,2,3,4,5]
A majority of the established protein structures have been determined by X-ray crystallography, because it is a well-established methodology for solving protein structures with a large span of molecular weights [2,6,7]
It is required to experimentally obtain high-quality protein crystals for the X-ray diffraction experiments, which is proven to be the bottleneck for many proteins [8,9], especially for those large proteins or protein complexes that are over 100 kDa [10,11,12]
Summary
X-ray crystallography has been the most powerful and robust approach to determining atomic protein structures, which provides incisive insights into the three-dimensional spatial arrangements of protein structures and allows for an in-depth exploration and understanding of the structure-related protein functions [1,2,3,4,5]. It is required to experimentally obtain high-quality protein crystals for the X-ray diffraction experiments, which is proven to be the bottleneck for many proteins [8,9], especially for those large proteins or protein complexes that are over 100 kDa [10,11,12]. Years of protein crystallographic studies have revealed that stable fragments or domains of proteins are more prone to crystallization, which increases the possibility of forming high-quality crystals for diffraction experiments [15,16]. The removal of flexible regions of protein molecules to create stable protein fragments and domains benefits developing crystallization-favoring crystals [17,18,19]. Digestion decreases the surface conformational entropy of protein surface residues, which is a critical indicator for successful protein crystallization [22,23,27,28]
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