Preparation of Sequence Modified Myoglobin Complexes and their Structural Analysis by NMR

Artificial proteins derived from sperm whale myoglobin shall be prepared by recombinant DNA-methodology at a laboratory scale. Through site directed mutagenesis of one amino acid residue a subset of the designed proteins have the ability to bind covalently to its native heme chromophore or (semi-)synthetic bile pigments. Additionally mini-myoglobin and micro-myoglobin with a significantly shorter amino acid sequence as compared to the native protein shall be prepared. These serve the purpose of precisely studying the binding properties with respect to structure and dynamics and - based on this - of improving the protein design with respect to selectivity and strength of binding. The final product of this design and preparation process is to be an artificial protein that can change its structure and properties reversibly under the influence of light, that is an artificial photo-receptor. The techniques use comprise among others - and apart from classical recombinant DNA technology - labeling with stable isotopes my molecular biology techniques for NMR structure determination and multi-dimensional homo- and hetero-nuclear NMR spectroscopy.

This project yielded important results in two areas: Protein science and methodological approaches for protein structure determination The most interesting new compound isolated is micro-myoglobin. Micro-myoglobin is to date the smalles known fragment of the muscle protein myoglobing, which still possesses the ability to bind the pigment heme, which causes the red color of bood and is responsible for oxygen transport. It was prepared by genetic engineering in bacteria an corresponds to the inner Exon of the myoglobin gene. Micromyoglobin fragment contains the minimum functionality required to bind heme. In the field of methodological advances novel methods of applying nuclear magnetic resonance (NMR) spectroscopy were developed. These methods were tailored for the investigation of substances containing free electrons, so-called paramagnetic substances, like myoglobin. The main innovation is that part of the interferences caused by the free electrons in magnetical investigations can be compensated. This is possible by exploiting a newly discovered effect PIN (paramagnetically induced narrowing). The determination of special arrangements (in particular angles) of parts of paramagnetic molecules becomes possible, in cases where this was not feasible previously.