Molecular cloning and protein folding characterization of a predicted heme-binding globin in a sugar cane EST database.

Abstract

A very large and representative sugar cane expression sequence tag (EST) library (SUCEST) was sequenced by a Brazilian consortium, opening the possibility to study important proteins, such as hemoglobins, which are largely present across the plant kingdom. The widespread presence and long evolutionary history of plant hemoglobins suggest a major role for this protein family in plants; however, little is known about their functional roles. In this study, we report the identification and characterization of a putative non-symbiotic hemoglobin cDNA clone that was identified in SUCEST. The cDNA was cloned, and the recombinant protein was purified and folded, as shown by its circular dichroism and emission fluorescence spectra. The expressed globin protein was able to bind hemin, as a characteristic Soret band was observed in the absorbance spectrum and increases were seen in the amount of secondary structure and in the stability of the protein. A model for the structure of the sugarcane hemoglobin was created using the crystal structure of a rice hemoglobin, and this model showed a conserved globular conformation.