Proteins are large molecules consisting of hundreds or thousands of amino acid residues forming a polymer with the amino acids arranged in a defined sequence. They are pivotal for all living organisms and of central importance for health care and biotechnology. Protein molecules exhibit distinct three- dimensional structural features governing their function. However, it is not entirely clear how the amino acid sequence determines structural features. In particular, this holds for the "hydrophobic effect" which arises because some amino acids with 'oil like properties' are quite insoluble in water. As a result, these residues tend to form an interior core of the protein molecule (alike a tiny oil droplet in water) to minimize exposure to the surrounding water. The incomplete understanding of the hydrophobic effect results from the enormous complexity of protein-water interactions. In turn, this prevents accurate prediction of structure and stability from sequence and accurate design of novel proteins. A primary potential benefit of this project will be a significant enhancement of the understanding of the hydrophobic effect. This will be accomplished by combining cutting-edge biophysics with computational protein design methodology in new and integrated protocols to enable more accurate protein structure prediction and engineering. All protocols and products of this project will be made publicly available. Excellent training of young scientists, in particular from underrepresented minorities, in protein biophysics and computational design, is a major goal of this project. Apart from highly individual mentoring of research and career development, annual summer schools will be held to reach communities outside of biophysical research and to increase interest in "STEM" in general.
It is a central conjecture of protein thermodynamics that the structured state of a protein as well as the loss of protein structure at very low temperatures ('cold denaturation') result from the hydrophobic effect. This project will focus on the engineering of cold denaturing proteins by iterating between computational design of protein structure and thermodynamic profile using the program Rosetta and biophysical characterization of the designs. The thermodynamic characterization will be pursued by use of circular dichroism (CD)-based Gibbs-Helmholtz analyses as well as differential scanning calorimetry (DSC), and the structural characterization of structured and denatured states by use of CD and nuclear magnetic resonance (NMR) spectroscopy, and small angle X-ray scattering (SAXS). Such iterative protein engineering will test and improve in an unprecedented manner our understanding of protein thermodynamics and computational design methodology.
