title: Monitoring of protein conformation by high-performance size-exclusion liquid chromatography and scanning diode array second-derivative UV absorption spectroscopy.
abstract: Genetic methods now allow the rapid production of mutant proteins for structure-function analysis. To properly interpret any change in biologic activity resulting from modification in primary sequence, it is essential to monitor conformational changes resulting from mutations. Several methods allow low-resolution protein conformational analysis. One method, second-derivative UV absorption spectroscopy, is particularly useful for proteins containing tyrosine and/or tryptophan residues. Using high-performance size-exclusion liquid chromatography and scanning diode array detection we have demonstrated that it is possible to monitor the degree of aggregation as well as conformational perturbation for a series of interleukin-2 structural mutants. Furthermore, the combination of high-performance liquid chromatography and second-derivative UV absorption spectroscopy avoids a potential artifactual contribution in non-chromatographic analysis due to protein aggregation.
authors.affiliation: Department of Pharmacology, Dartmouth Medical School, Hanover, NH 03756.
authors.forename: C E
authors.initials: CE
authors.lastname: Ackland
authors.forename: W G
authors.initials: WG
authors.lastname: Berndt
authors.forename: J E
authors.initials: JE
authors.lastname: Frezza
authors.forename: B E
authors.initials: BE
authors.lastname: Landgraf
authors.forename: K W
authors.initials: KW
authors.lastname: Pritchard
authors.forename: T L
authors.initials: TL
authors.lastname: Ciardelli
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007376:Interleukin-2 / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
chemical_list: D007376:Interleukin-2