Structure and Dynamics in the Folding Process of ACBP

The mechanism of how a linear protein synthesized on the ribosome achieves its native state in a reliable and efficient way is still one of the most intriguing problems in structural chemistry. The topic of protein folding has gained in importance since the complete decoding of the human genome in the year 2001. Thousands of protein products of the identified genes are of unknown structure and function, which could possibly be elucidated by a sequence-based method of fold prediction. On the other hand, evidence has been accumulated that partially or misfolded states of proteins are the basis of diseases like Kreutzfeld-Jakob disease, Alzheimer`s disease, or Parkinson`s disease. The main interest of this project is the investigation of molecular determinants in the protein folding process. As model system the acyl-coenzyme A binding protein (ACBP) will be used. The project represents a comparative study of bovine ACBP and its related homologue from yeast. Several different methods based on the technique of nuclear magnetic resonance spectroscopy (NMR) shall provide complementary information about structure, dynamics, and kinetics of these proteins during the folding reactions. The main points of emphasis will be on the one hand a thorough analysis of the denatured states of bovine and yeast ACBP, which possibly possess crucial structural features of the respective native states. Chemical shift analysis of the backbone atoms and relaxation studies of the backbone amide 15N nuclei will indicate the content of residual secondary structure and conformational flexibility. The effect of spin-label induced relaxation provides information about the three- dimensional assembly of the highly flexible protein backbone in the denatured state. On the other hand, the kinetics of the folding reaction will be analyzed by a quenched-flow kinetic study with NMR detected hydrogen exchange in the yeast ACBP folding reaction in order to find the earliest transient intermediates on the way to the native fold. From an analysis of the primary structures of the known ACBP variants and a comparison to the obtained kinetic results, it is the hope to identify potential determinants in the amino acid sequence for the fast and efficient folding of ACBP. Additionally, investigation of the two forms of ACBP of two different species shall answer the question whether and to which extent folding properties of ACBP are conserved during evolution.