Biomolecular NMR

Three positions available for Ph.D. or Masters students at the Biomolecular NMR Laboratory

a) Structural and Biochemical characterization of novel Fe2+ metabolic pathways

b) Structural proteomics of thermostable protein libraries by NMR

c) Structural and biochemical investigation of cell wall formation in spores

Students will be trained in biochemical and biophysical methods in addition to advanced NMR methods. The choice to specialize in molecular biology and heterologous protein expression or in more computationally intensive tasks such as protein structure and dynamics determination will be available. The biological problems that will be investigated are: Protein structure elucidation and dynamics studies, Protein-protein interactions, Metal binding to proteins, paramagnetic NMR, study of large proteins and solid state NMR.

Structural and Biochemical characterization of novel Fe2+ metabolic pathways
The object of this project is to study the metabolic pathways of Iron in the ferrous state in bacteria. Novel operons that are thought to be related to ferrous iron transport has been identified in many separate genomes. Little is known about their structure and function apart from predictions based on bioinformatic tools. Some small proteins that are thought to transport Fe2+ have been proposed. Such types of iron transporters have not been adequately characterized in spite of the crucial role that iron has in almost every organism. Apart from the obvious scientific interest in such systems, it is also possible that pharmaceutical applications can be explored by targeting this metabolic pathway.

Structural proteomics of thermostable protein libraries by NMR 
In the framework of collaboration with the Protein Biochemistry, Folding and Stability Laboratory at ITQB we propose the proteomic based study of thermostable proteins. Interest in such proteins originated from the study of thermostable organisms that are known to thrive at extreme temperatures reaching 95 oC, well past the point where mesophylic (those living at ambient conditions) proteins denature. Recent protein screening methods have identified large numbers of previously unknown proteins that are over-expressed during thermal stress. The goal of the student in this project will be to identify, express and structurally characterize these proteins by NMR and biophysical methods. In the process of this project we aim to achieve the secondary goal of developing both manual and automated pathways for structure elucidation using NMR that will pave the way for a larger scale proteomics and functional proteomics initiative.

Structural and biochemical investigation of cell wall formation in spores 
In the framework of a collaboration with the Microbial Development Group at the ITQB, we also propose projects in the structural characterization of proteins of the surface of the bacterial spore. These are highly resilient cell types whose surface is covered by a multi-protein structure called the coat. In pathogenic species such as Bacillus cereus or B. anthracis, or in the model organism B. subtilis, the coat acts as a protective shield that contributes to the survival and persistence of spores. The coat structure results from the ordered assembly of over 70 different polypeptides. Some of the coat proteins have highly unusual primary sequences which may be related to their roles in spore protection but also with their mechanism of assembly. Recruitment of the coat proteins to the surface of the developing spore is in part controlled by specific protein-protein interactions and, once at the spore surface, some of these proteins will undergo extensive multimerization. We propose to conduct the structural characterization of abundant low molecular weight structural components of the spore coat, and of their complexes, in Bacillus subtilis. The work will involve the overproduction of selected proteins, both the wild type as well as mutated forms, and their purification for structural characterization by NMR spectroscopy.