Abstract
Preparative flat-bed gel isoelectric focusing has reduced or eliminated observed heterogeneity in three different proteins, thereby greatly improving the quality of crystals grown from each of them. This improvement is manifested both in crystal size and in increased measurability of diffraction data. Isoelectric focusing is a general and nondestructive technique that may facilitate the crystallographic study of proteins which are biologically interesting, but which, for reasons of microheterogeneity, do not yield diffraction quality crystals.
Highlights
Naval Research Laboratory, Washington,D.C. 20375 phosphorylation, and methylation, may not be complete and the moieties attached may be heterogeneous as well
Along the lines of the fist approach, McPherson [1]and Blundell and Johnson [2] have summarized successful crystallization procedures up to 1976 and McPherson has demonstrated the merits of polyethylene glycols as precipitants [3].Carter and Carte(r4) havedescribed the use of incomplete factorial experiments for investigating crystallization conditions where time and materials are in limited supply
In this paper,we explore the utility of preparative flat-bedgel isoelectric focusing to achieve higher purity in the starting material and report the success of the technique in improving the quality of crystals of three separateproteins
Summary
Naval Research Laboratory, Washington,D.C. 20375 phosphorylation, and methylation, may not be complete and the moieties attached may be heterogeneous as well. Protein crystallization depends upon specific weak attractive interactions between protein molecules which are repeated over Preparative flat-bed gel isoelectric focusing has re- many thousands of molecules to form an ordered three-dimmddituiyefecfnaoeestfrduiecsornrramtybesaplitilnarmitoilfsyitenegsairotnteofesdw,ddntobihbffoefrsrtroehaemrbcvtiyenieodagnhccrerhetdyaesatortltfoyaaglt.iehmsneIiesmzpoiert.eyolaTvefnichnotdircgisniucitisnmhtihnepirgnreqicoesurvaeela-mpta-hsrreeeoendtiqesnuiitonietnriesaalcsinptliaecotcontiinfcsitecua..rcfMWtawciheciistrlh,oechomoenutolelysdrtosegooaelfsvnitelehyniettty,est,rhumiferfiiiantncatoeetcerocacfucrrtayisnscatgratylsosugtrrrafolnalweicnaetehrs a generaland nondestructive technique that mafyacil- at thatpoint or cause serious lattice perturbations. Itate the crystallographic study of proteins which are Microheterogeneity is usually undetected when only biobiologicallyinteresting, buwt hichf,or reasons of chemical activity is monitored during protein isolation and is microheterogeneity, donotyielddiffractionquality difficult to detect when gel filtration and sodium dodecyl crystals. Protein crystallographers are addressing specific biochemical questions which require the study of specific proteins and present increasingly challenging crystallization problems. Two approaches to overcoming these problems are the development of more systematic means to find crystallization conditions and theimplementation of methods to increase the homogeneity of the starting material. Shore and Feher [5] have reported a light scattering method to predict whethersmallprotein aggregates willgrow to crystals or form amorphous precipitate.several work-
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