Crystal Structure of Ser-22/Ile-25 Form Crambin Confirms Solvent, Side Chain Substate Correlations

Abstract

Motion correlated over 5-8 Å (liquid-like movement) has been shown by the non-Bragg technique of x-ray diffuse scattering to be important in insulin and lysozyme crystals (1, 2). State of the art molecular dynamics methods cannot model such correlations (3), perhaps because of inadequate sampling of conformational substates (4). Multiple substates of nearly equal energy are also proposed for myoglobin based on spectroscopic evidence (5-7), but spectroscopy is not well suited to elucidate the nature of these substates. Neither is NMR, unless extremely tight distance restraints are used (8).Diffraction from a crystal is averaged over many unit cells and over the time spent on data collection. It is generally believed that this averaging precludes extracting dynamic information, such as occurrence of multiple substate correlations from an x-ray structure. However, nonrandom correlations will contribute to Bragg reflections. Given diffraction data beyond 1.4 Å (9), the correlations can be modeled as substate disorder and provide insight into protein dynamics. If one could physically separate the substates and study each separately, one could prove such correlations exist and derive the rules for the correlation.Crambin presents an excellent system for such an experiment. Crambin from the natural source contains two sequence isomers in a 3:2 ratio (10, 11), the so-called mixed form of crambin. The major isomer has Pro and Leu at positions 22 and 25, respectively (the PL form); 1 the minor isomer has Ser and Ile at the same positions (the SI form). In the mixed form crystal structure, side chain electron densities for heterogeneous residues are superimposed (Pro and Ser at residue 22 and Leu and Ile at residue 25). The Tyr-29 side chain from a 2 1 -screw axis-related molecule has close contacts with the Pro or Ser residue and adopts two conformations. A proposed correlation of the Tyr-29 conformation with the identity of the amino acid at residue 22 (12) has been supported by the PL form structure (13). Now the second or SI form of crambin has been purified by fast protein liquid chromatography and crystallized. It establishes the side chain correlations definitively and establishes associated solvent interactions.In this paper, first the proposed mixed form protein networks are extended to water disorder using stereochemical "rules," such as van der Waals' contacts and hydrogen bonding. Second, these postulated networks are compared with the crystal structures of the physically separated pure forms of crambin: the PL form structure and the newly determined SI form structure. These results establish that the x-ray structure of the mixed form of crambin can elucidate substate spatial correlations between side chains and solvent, as proven by the pure form structures.Alternative conformations at disordered residues and water molecules are designated by attaching A and B to the residue number. Such disordered conformations are often correlated with neighboring residue disorder through space and may represent conformation...

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