Molecular Biology: Protein Structure and Function - Stepanov V.M. 2005

Tertiary protein structure
Features of X-ray diffraction analysis as the primary source of information on protein spatial structure

X-ray crystallographic Analysis of Protein crystals remains the primary METHOD FOR DETERMINING their three-dimensional Structure. Recently, nuclear magnetic Resonance (NMR) spectroscopy has also emerged as a powerful tool for studying Cell/13.html">Protein Structure in solution; however, despite their immense value, these studies are currently limited to relatively small Proteins.

Without delving into the technical details of X-ray crystallography, let us briefly examine its potential limitations. Since X-ray analysis investigates The structure of a protein molecule within a crystal, a crucial question arises: how accurately does the state of the molecule in the crystal reflect its structure—and consequently its behavior—in solution, which is essential for its biological function? At first glance, intermolecular interactions between protein globules that inevitably occur during crystallization might significantly distort the protein structure, thereby undermining the method's reliability. However, numerous observations have shown that this is not the case.

This Conclusion is supported, first, by the fact that NMR data on the three-dimensional structure of proteins in solution are consistent with X-ray crystallography results. Second, catalytic activity has been observed in the crystals of certain Enzymes, indicating that the conformation characteristic of the native protein is preserved in the crystal state. Moreover, soaking crystals in a solution of a substrate (or, more commonly, its analogue)—a standard Procedure in Protein Crystallography—leads to substrate binding at the Active Site, further confirming the retention of the native structure within the crystal. Third, for proteins whose crystals contain molecules with different orientations, these molecules possess virtually identical three-dimensional structures despite differences in crystal contacts with neighboring molecules, if we disregard minor positional variations of functional groups at the contact interfaces.

It is worth noting that conformational changes or the Selection of a specific conformation during the crystallization of small Organic compounds are fairly common occurrences. In contrast, the preservation of conformation during protein crystallization is due to the unique structural properties of protein crystals. Protein crystals invariably contain a large volume of Water and buffer salts—the crystallization solvent—with the solvent fraction often reaching 50% or more of the total volume. Consequently, although a protein molecule in a crystal forms numerous non-covalent contacts with neighboring molecules, it remains immersed in essentially the same environment it experienced prior to crystallization. In particular, researchers can detect a layer of several hundred water molecules directly bound to the protein surface, forming its Hydration shell. Thus, owing to the unique nature of protein crystals, their formation does not induce major structural alterations in the protein, and X-ray crystallographic analysis generally provides an accurate representation of the protein molecule's structure in solution.

Nevertheless, one must account for a significant limitation: the internal dynamics of the protein structure within a crystal are constrained by contacts with adjacent molecules. This is especially true for large-scale structural rearrangements; therefore, one should be cautious not to underestimate the dynamic potential of proteins when relying solely on the findings of crystallographic studies.

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