Fig 1.
Model for competitive dimerization.
(A) Sequence alignments of LOV proteins in Methylocystis compared to fungal analogues and the LOV 2 domain of Avena sativa Phototropin (AsLOV2). Comparisons of a LOV-transcription factor in Methylocystis (McLOV-TF) and Methylocystis rosea (McLOVr-TF) demonstrate high homology to sLOV proteins in the respective organisms (McLOVn, McLOVr). Key elements required for signal transduction are conserved in the fungal sLOV proteins Trichoderma reesei ENVOY (ENV1) and Neurospora crassa Vivid (VVD) as well as fungal LOV transcription factors N. crassa White-Collar 1 (WC1) and T. reesei Blue-light receptor 1 (BLR1). These elements are not conserved in AsLOV2 or short LOV domains from P. putida (PpLOV) or R. sphaeroides (RsLOV). Specifically, a key hinge region is conserved in all bacterial and fugal proteins (light green) within the NCap. Two additional residues are absolutely conserved that form key contacts in organization of the NCap hinge region. Core signaling regions of the LOV domain (blue) are conserved in all species. Residues absolutely conserved in all species (*) are noted, residues conserved in fungal and bacterial species, but not AsLOV2 are shown (^) as well as strongly conserved elements (:). (B) Domain architecture in Methylocystis LOV proteins. (C) SEC of McLOVr (black), McLOVn (green) and 337-TF (blue). All elute as dimers with apparent molecular weights (MW) of 35 kDa (McLOVr), 33.5 kDa (McLOVn) and 32 kDa (337-TF). The expected MW of a monomer was 17 kDa. Some preparations of McLOVn contain a significant monomeric fraction. Two distinct peaks with apparent MWs of 34 kDa and 17 kDa are observed.
Fig 2.
McLOV photocycles and kinetics.
(A-C) McLOV proteins all demonstrate spectra consistent with C4a adduct formation. Dark-state spectra (black) are consistent with oxidized flavin. Illumination with blue light bleaches the 450 nm absorption bands (red). Subtle differences in degrees of photoactivation in McLOVn (A), 337-TF (B) and McLOVr (C) are indicated by residual presence of absorption bands centered around 450 with vibrational bands at 425 and 478 nm. (D-F) McLOVn (D), 337-TF (E) demonstrate first order kinetics as demonstrated by an absorbance trace at 450 nm and their respective residual plots. In contrast McLOVr (F) is best fit with a biexponential function.
Table 1.
Kinetics of thermal reversion for LOV constructs and variants at 296 K.
Fig 3.
Arrhenius and Eyring analysis of McLOV proteins.
Arrhenius (A,C,E) and Eyring (B,D,F) of McLOVn (A,B), 337-TF (C,D), and McLOVr (E,F) constructs. All measurements were carried out in triplicate, and error bars are shown as standard deviations relative to the mean. McLOVn and McLOVr both depict weak temperature dependence that indicates low entropies of activation with entropic compensation. In contrast 337-TF, has markedly increased enthalpies of activation (71 kJ/mole vs. ~50 KJ/mole) with a decrease in the entropic penalty (-44 J/mole*K vs. ~ -100 J/mole*K).
Fig 4.
External bases do not catalyze some McLOV proteins.
(A,B) Both McLOVn (A) and McLOVr (B) show no dependence of adduct decay on imidazole concentration. In contrast, a Thr27Ile demonstrates spectra consistent with LOV chemistry (E) with a 100-fold decrease in the rate of adduct decay (F). The Thr27Ile variant causes an imidazole concentration dependent rate of adduct scission (C). Thus, Thr27 renders McLOV proteins insensitive to imidazole catalysis. (D) In contrast, 337-TF is imidazole sensitive despite containing a Thr at a position equivalent to 27. (G) A LOV structure (modeled from ENVOY PDB: 4WUJ) containing a Thr at a position equivalent to 27. Thr27 can coordinate a hypothetical water molecule between the N5 position on flavin, C61 and Q124 (McLOVr numbering). In all figures, error bars represent the standard deviation of the mean for experiments conducted in triplicate.