Mass instability in isolated recombinant FixL heme domains of Bradyrhizobium japonicum.
Several recombinant Bradyrhizobium japonicum FixL heme domains (BjFixLH) have been characterized and their temporal mass stabilities assessed by MALDI-TOF mass spectrometry. The intact heme domains all bound heme and gave normal UV-visible spectra, indicating that they were correctly assembled. Proteins produced at Washington State University included a parent 131-amino acid "full-length heme domain" (FLHD) of primary sequence T140-Q270 (BjFixLH140-270), a histidine-tagged analogue containing an N-terminal extension, and five different terminus-truncated variants. The smallest of these was a 106-amino acid "core PAS heme domain" with primary sequence T151-L256. All variants except for the smallest exhibited significant mass instability, assessed by MALDI-TOF mass spectrometry, that was apparent within 1-16 days standing in a sterile environment at room temperature. Two full-length heme domains expressed independently in geographically remote laboratories (Northern Illinois University and JILA, University of Colorado) also exhibited this mass instability. A mass loss of as much as approximately 25% of the starting mass has been observed, which could explain the "missing" terminal amino acids in published crystal structures. This work documents the phenomenon and its persistence despite (i) sample sterilization, (ii) protease inhibitors, (iii) primary sequence variations, (iv) the presence or absence of ferriheme ligands, and (v) the presence or absence of O2.
Satterlee
James D
JD
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA. hemeteam@wsu.edu
Suquet
Christine
C
Bidwai
Anil K
AK
Erman
James E
JE
Schwall
Linda
L
Jimenez
Ralph
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001483:Base Sequence; D020369:Bradyrhizobium / Q000737:chemistry*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D000071677:Histidine Kinase; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D017931:DNA Primers; D006420:Hemeproteins; D011994:Recombinant Proteins; D006418:Heme; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase