title: Can protein conformers be fractionated by crystallization?
abstract: Molecular crystallization typically singles out a specific conformation, or a set of conformations that are identical over large parts and may show some flexibility, from a mixture of equilibrating conformations in solution. To critically evaluate the selectivity of this process, human lactate dehydrogenase isozyme 1 (LDH-1) microcrystals were separately dissolved and subsequently assayed inside capillaries with electrophoretically mediated microanalysis (EMMA) at both the ensemble and the single-molecule level. While fragments from the same crystal exhibited identical enzyme activities, different crystals, even when grown from the same drop of mother liquor, showed markedly different activities. Activities of individual molecules from a crystal were found to be essentially identical, whereas molecules obtained directly from solution showed a 4-fold variation in activity. Furthermore, after storage at 37 °C, the distribution of single-molecule LDH activities from solutions of individual crystals broadened and approached that of LDH obtained from the original solution. X-ray crystallography also showed distinct conformations for single microcrystals and confirms that crystallization properly selects even small conformational variants of proteins and that the slow equilibration to multiple stable conformations in solution is responsible for the observed single-molecule heterogeneity.
authors.affiliation: Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
authors.forename: Aoshuang
authors.initials: A
authors.lastname: Xu
authors.forename: Fenglei
authors.initials: F
authors.lastname: Li
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.forename: Edward S
authors.initials: ES
authors.lastname: Yeung
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; C409667:lactate dehydrogenase 1