Characterization of the truncated hemoglobin THB1 from protein extracts of Chlamydomonas reinhardtii

Abstract

Truncated hemoglobins (TrHbs) belong to the hemoglobin superfamily, but unlike their distant vertebrate relatives, little is known about their principal physiologic functions. Several TrHbs have been studied in vitro using engineered recombinant peptides. These efforts have resulted in a wealth of knowledge about the chemical properties of TrHbs and have generated interesting functional leads. However, questions persist as to how closely these engineered proteins mimic their counterparts within the native cell. In this report, we examined THB1, one of several TrHbs from the model organism Chlamydomonas reinhardtii. The recombinant THB1 (rTHB1) has favorable solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the in vivo protein is a critical step in understanding the physiologic function of this protein. Using a simplified three-step purification protocol, 3.5-L batches of algal culture were processed to isolate 50–60 μL fractions enriched in THB1. These fractions of C. reinhardtii proteins were then subjected to physical examination. Using gel mobility, optical absorbance and immunoreactivity, THB1 was identified in these enriched fractions and its presence correlated with that of a heme molecule. Mass spectrometry confirmed this cofactor to be a type b heme and revealed that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage.

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

“…These proteins were obtained in monodisperse form and confirmed the origin of the multiple masses in the recombinant WT preparation. At this time, it is not known if the terminal methionine is cleaved in the C. reinhardtii cell, and whether the terminal residue is acetylated as observed for THB1 [18]. We were unable to attribute specific consequences to N-terminus heterogeneity and used the WT, P3A and S2_P3insA proteins interchangeably.…”

Dynamics of Lysine as a Heme Axial Ligand: NMR Analysis of the Chlamydomonas reinhardtii Hemoglobin THB1

“…Mass spectrometry of the purified WT protein confirmed that Met1 is completely cleaved when THB1 is isolated from Escherichia coli. Native THB1 extracted from C. reinhardtii cells is N-acetylated, 33 a posttranslational modification that does not occur in the heterologous expression procedure. However, the N-terminus of the protein is disordered, 22 and the modification is not expected to influence the heme cavity.…”

Production of Natural Pigments Using Microorganisms

“…It is noteworthy that microalgae are classified as both microorganisms and lower plants, and they play a significant role in the production of natural pigments through fermentation and genetic engineering. Examples include Chlamydomonas reinhardtii for hemoglobin, Haematococcus pluvialis and Chromochloris zofingiensis for astaxanthin, , Galdieria sulphuraria for phycocyanin, Phaeodactylum tricornutum for fucoxanthin, and Chlorella protothecoides for lutein production …”