Origins of aging mass loss in recombinant N-terminus and C-terminus deletion mutants of the heme-PAS biosensor domain BjFixLH140–270

Nine recombinant FixL heme domains from Bradyrhizobium japonicum previously were shown to exhibit mass instability independent of many environmental factors (J.D. Satterlee, C. Suquet, A. Bidwai, J. Erman, L. Schwall, R. Jimenez, Biochemistry 47 (2008) 1540–1553). Two of those recombinant proteins were produced in remote laboratories. Mass losses begin appearing at completion of isolation and comprise a substantial proportion of samples within 1–3 days of storage and handling. Thus, degradation occurs during the time frame of experiments and crystallization. Detailed understanding of this instability is desired in order to formulate stable heme-PAS sensor domains for experimentation and for a mechanistic interpretation. However, mass spectra of the full length heme-PAS domain, BjFixLH140–270, are complex by 1–3 days following isolation due to broad features and a high density of overlapping peaks, so that individual peak assignments are at present ambiguous. This stymies direct, quantitative interpretation of the source of the observed mass losses. To solve this dilemma amino-terminal primary sequencing and MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry monitoring of three terminal variants of BjFixLH140–270 have been achieved. The working hypothesis, that the experimentally observed mass losses originate in the PAS protein sequence termini, has been substantiated. This establishes a basis for interpreting the more complex results from aging full length BjFixLH140–270.

Graphical AbstractMass spectrometry and primary sequencing reveal that the origin of observed mass degradation in heme biosensor domains, BjFixLH140–270, is loss of amino acids at both the N-terminus and C-terminus of the protein.Figure optionsDownload as PowerPoint slide