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The Effect of Glutamine Insertions on the Structure and Function of gMDH
Author(s) -
Chikowski Rachel,
Bell Ellis,
Bell Jessica
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.851.4
Subject(s) - glutamine , mutant , crystal structure , chemistry , wild type , protein crystallization , crystallography , biochemistry , biology , crystallization , amino acid , organic chemistry , gene
Extended polyglutamine (polyQ) stretches in proteins are known to cause neurodegenerative disease such as Huntington's disease and spinocerebellar ataxia type 3, which are extremely pathogenic and often fatal. Protein misfolding and aggregation are characteristic of polyQ expansion diseases. This study explores the effect of glutamine insertions on glyoxysomal malate dehydrogenase (gMDH) structure and function. Two mutants, 240Q10 and 301Q10, each containing a ten glutamine insert were constructed. Insertion of glutamines and purity were assessed using MALDI‐TOF mass spectrometry. Enzymatic activity of each mutant was compared to native gMDH. Results show decreases in Vmax for both mutants as compared to native, but more drastic decreases for mutant 240Q10. Km values for substrates showed minimal changes. Crystals of 301Q10 were grown under conditions similar to native gMDH. A 2.5 angstrom dataset was collected with a final Rmerge of 0.118, space group P21 (a=90.57, b,c=95.68 angstrom and ?уz ?~z ? ?=90 ?X). The structure was solved by molecular replacement using native gMDH as the probe. Model building and refinement are in progress. Although at this stage in the structural refinement the 10 glutamine insert is not resolved, this mutant¡¦ s crystal lattice packing differs from native gMDH. This research is supported by a grant from the National Science Foundation: MCB 0448905 to EB.