The long-term objective of this project is to develop the first experimental tool capable of quantitatively measuring the conformation of heme in solution. This tool will be an important contribution to the structural biology initiative of the NIH roadmap, which could be used in future studies that determine the nuclear magnetic resonance (NMR) solution structure of heme proteins. Heme proteins have a diverse set of biological functions, including: electron transport, gas transport, small molecule sensing, and catalysis. Each heme protein optimizes the reactivity of the heme cofactor to achieve its specific function and heme conformation is arguably the most under-investigated strategy available. It is essential to understand this determinant of heme electronic structure and reactivity to fully comprehend the biochemical processes involving heme proteins, and diseases associated with their malfunction. The cytochrome c family of proteins will be used as a model system to develop this tool because this family utilizes a nonplanar heme cofactor and the protein matrix provides a strategy to alter the heme conformation in a systematic way that is unlikely to introduce large-scale protein conformational changes. Paramagnetic 1H and 13C NMR spectroscopy will be used as a spectroscopic probe because this technique can simultaneously examine the electronic structure of heme at several positions on the porphyrin macrocycle. Multi-dimensional NMR experiments and selective isotopic enrichment of the heme cofactor will be used to make heme paramagnetic 1H and 13C resonance assignments. Density functional theory (DFT) calculations, initially assisted by X-ray crystallography and resonance Raman spectroscopy, will be used to interpret the paramagnetic NMR data in terms of out-of-plane distortions of the heme cofactor. Ultimately, this strategy will produce a DFT-based correlation between paramagnetic NMR data and heme conformation. This new tool can be applied to a wide range of heme proteins that have important functions within biological systems. The DFT-based correlation will also identify the relationship between heme conformation, electronic structure, and reactivity.
Biological organisms, including humans, use heme to carry out a diverse set of fundamental processes. To maximize the efficiency of a specific process, biological systems manipulate heme and its surroundings. Arguably, the most under-investigated manipulation available to heme proteins is changing the shape, and this proposal will develop the first method to measure the shape of heme in solution.
| Josephs, Tracy M; Liptak, Matthew D; Hughes, Gillian et al. (2013) Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination. J Biol Inorg Chem 18:289-97 |
| Liptak, Matthew D; Fagerlund, Robert D; Ledgerwood, Elizabeth C et al. (2011) The proapoptotic G41S mutation to human cytochrome c alters the heme electronic structure and increases the electron self-exchange rate. J Am Chem Soc 133:1153-5 |
| Liptak, Matthew D; Wen, Xin; Bren, Kara L (2010) NMR and DFT investigation of heme ruffling: functional implications for cytochrome c. J Am Chem Soc 132:9753-63 |
