Monitoring of protein conformation by high-performance size-exclusion liquid chromatography and scanning diode array second-derivative UV absorption spectroscopy.
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.
Ackland
C E
CE
Department of Pharmacology, Dartmouth Medical School, Hanover, NH 03756.
Berndt
W G
WG
Frezza
J E
JE
Landgraf
B E
BE
Pritchard
K W
KW
Ciardelli
T L
TL
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*
D007376:Interleukin-2