John Markley became emeritus at the end of May, 2020. He has grants that enable him to maintain ongoing research projects, which he directs remotely from his home in Colorado.
The central theme of our research is the application of nuclear magnetic resonance (NMR) spectroscopy to the solution of biochemical problems. The unique power of NMR lies in its ability to provide detailed chemical and structural information at an atomic level about molecules in solution — even when they are present in living cells or organisms.
The general strategy is to use multidimensional (2D, 3D, and 4D) multinuclear magnetic resonance techniques to detect and assign resonances from atoms of biological interest (e.g. 1H, 13C, 15N, and 31P). With these assignments in hand, we can then interpret the wealth of spectral information present in coupling constants, relaxation rates, cross-relaxation rates, and chemical shifts. Proton-proton cross-relaxation rates and a variety of measured coupling constants are used to derive three-dimensional structures of these macromolecules. Relaxation rates, line-shapes, and Nuclear Overhauser Effect measurements provide information about molecular motions and conformational changes. The kinds of information gained from such investigations can be critical for learning how these molecules work and how they can be redesigned to have desired properties.
We exploit recombinant DNA technology as a means for producing the large amounts of protein needed for NMR investigations and for introducing stable isotopes of interest (most commonly 2H, 13C, and 15N). Mutagenesis studies allow us to test hypotheses about the roles of individual amino acid residues in determining properties such as local structure, conformations and mobilities of side chains, hydrogen exchange kinetics, rates of protein folding or unfolding, pKa values, oxidation-reduction potentials, and ligand binding.
In particular, our work focuses on the following topics:
(i) Modulation of iron-sulfur cluster properties
Iron-sulfur proteins participate in many biochemical processes including electron transfer, substrate binding and catalysis, and regulation and sensing. As their name implies, these metalloproteins contain one or more iron ions ligated mostly, though not exclusively, by sulfur atoms. The immediate environment of the metal ion(s) has a significant impact on its properties but the details of this phenomenon are poorly understood. Through studies on Clostridium pasteurianum rubredoxin and selected [2Fe2S] ferredoxins we hope to advance our understanding of how sequence and structure determine the properties of iron ions in iron-sulfur proteins.
(ii) Brazzein – a sweet-tasting protein
Found in a variety of African and South Asian fruits, sweet-tasting proteins are perceived sweet by humans and old-world monkeys. In the last 30 years six such proteins have been discovered — they all have different molecular lengths, as well as very little sequence and structural homology. Despite significant research efforts, the features responsible for the sweet taste of these proteins are still very poorly understood. Our work focuses on brazzein, a sweet protein of 54 amino acid residues, and its interaction with sweet taste receptors.
(iii) Acyl carrier protein
Acyl carrier protein is a small protein involved in the movement of various substrates between enzymes in biosynthetic pathways. One of its functions involves carrying unsaturated long chain fatty acids to the enzyme delta-9-desaturase which introduces a double bond between C9 and C10 of the fatty acid chain. Work in the lab concentrates on the structural characterization of acyl carrier protein. Significant effort is also devoted to advance our understanding on how acyl carrier protein binds to delta-9-desaturase.
(iv) Biogenesis of iron-sulfur clusters
Iron-sulfur clusters are ubiquitous in nature and participate in many biochemical processes. As incorrect cluster assembly can result in disease, it is imperative portant to understand the mechanism behind steps leading to such problems. We study three proteins involved in the biogenesis of iron-sulfur clusters: IscU, HscA and HscB. Available experimental evidence strongly suggests that IscU acts as a scaffold for the assembly and subsequent transfer of Fe/S clusters to target apo-proteins. The details of these steps, however as well as the roles of HscA and HscB in the cluster transfer process are not well understood. We are using NMR spectroscopy to gain novel insight into these unexplored areas of Fe/S biochemistry.
(v) Automation in NMR investigation of protein structure
Alternately, graduate students and postdoctoral fellows in the laboratory may focus on developing instrumentation or novel ways of collecting or analyzing NMR data. Current developed software and techniques in our laboratory include:
– PISTACHIO: A probabilistic approach to chemical shift assignment
– HIFI-NMR: Fast data collection and processing of multidimensional NMR
– PECAN: Secondary structure prediction of proteins
– LACS: Detection of outlier chemical shifts
– ALMONDS: Chemical shift prediction based on sequence