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2-Methyl-2,4-pentanediol (MPD) is the most popular chemical additive used for crystallization of biological macromolecules. However, the mechanism of its action on proteins in aqueous solution is not well understood. We have carried out a systematic analysis of the conformation and environment of MPD molecules bound to proteins. We find that the majority of MPD molecules adopt their most stable conformer. They prefer to bind to hydrophobic sites with a distinct preference for leucine side chains. Most MPD binding sites involve amino-acid residues in helical or β-sheet structures. MPD binding to proteins is penetrative, leading to displacement of water molecules in grooves and cavities (sometimes ligand-binding and active sites) on the protein surface. This results in a large reduction of solvent-accessible area, which can have significant implications for protein stability. The packing of the MPD molecules by the protein is not optimal and usually some other solvent molecules are also bound along with it. Our analysis suggests that MPD is not as strong a denaturant as often suggested. It promotes stabilization of the protein by preferential hydration, which is facilitated by attachment of MPD molecules to the hydrophobic surface.
research papers
Pokeweed antiviral protein, PAP-Saci, isolated from seeds of the Chinese pokeweed plant, Phytolacca acinosa, belongs to the family of type-1 ribosome-inactivating proteins (RIPs). Type-1 RIPs are ~30-kDa N-glycosidases that inactivate eukaryotic and prokaryotic ribosomes via a site-specific depurination of ribosomal RNA (rRNA). Here we describe the preliminary X-ray structure determination at 1.7 Å resolution of one PAP isoenzyme from seeds, PAP-S1aci, after crystallisation from a heterogeneous mixture of two isoenzymes. PAP-S1aci possesses a rare type of glycosylation, specifically, N-linked N-acetyl-D-glucosamine monosaccharide (GlcNAc) substitutions at canonical Asn-Xaa-Ser/Thr sequons. One GlcNAc residue was found to play a critical role in crystal lattice formation, forming a packing interface across a crystallographic two-fold with the identical sequon of an adjacent monomer. This observation suggests that deglycosylation protocols for the crystallisation of glycoproteins should be designed to allow for exploitation of the crystal packing potential of the innermost core sugar residue (N-linked GlcNAc or O-linked GalNAc).
