Research

Conformational Dynamics of Biomolecules

In solution, biological macromolecules like proteins and nucleic acids are not rigid but populate a range of conformations. Some of these alternate conformations can have significantly different structures involving changes in the secondary and tertiary structure of the protein or RNA. Not surprisingly these alternate conformations can have very different properties like enzymatic activities, binding constants etc. As these alternate conformational states have low populations and lifetimes varying from microseconds to hundreds of milliseconds they cannot be detected by conventional NMR experiments. Recently we have developed NMR experiments to obtain structures of excited states with lifetimes ranging from 50 μs to 50 milliseconds [1-3]. We use NMR spectroscopy and combine it with computational and fluorescence techniques to obtain an atomic resolution picture of the dynamics of biomolecules [4-5]. Computational methods [5] and fluorescence techniques provide complementary information to that obtained by NMR methods and we plan to use these techniques in collaboration with our colleagues. Students joining the lab will learn state of the art solution NMR methods, biochemistry/molecular biology methods and computational & fluorescence techniques in collaboration with experts. Specific projects that we are working on are listed below.

Conformational dynamics of T4 lysozyme L99A

T4 Lysozyme L99A interconverts between two compact forms (Fig. 1A) on the millisecond timescale [4]. The structure of the minor form populated to only 3% has been obtained using CPMG NMR experiments. However several questions remain unanswered. For example: What is the height of the barrier? What provides the energy for this barrier-crossing event? We are investigating these using a combination of NMR, computational (with Jagganath Mondal) and mutagenesis techniques.

Folding of knotted proteins and multidomain proteins

Knotted proteins essentially contain a knot. However their folding mechanism is largely unknown. Similarly little is known about the detailed folding mechanism of multidomain proteins. In collaboration with Kanchan Garai we are trying to understand the folding of apolipoprotein E a multidomain protein that is risk factor in Alzheimers. We are using NMR/Fluorescence techniques to investigate the folding mechanism of these proteins.

Conformational changes in large RNA molecules

Just like proteins, RNA molecules undergo large conformational changes. For example the conformation of riboswitches can change in response to a ligand or temperature and regulate the expression of proteins. These conformational transitions can affect the secondary structure not just the tertiary structures. We are currently working on the 56 nt P5ABC domain which undergoes change in 2o structure upon binding Mg2+ and the spliced leader sequence from L collosma that interconverts between two forms on the 100 ms timescale.

Development of new NMR experiments

Despite the development of powerful new relaxation dispersion NMR methods to study the conformational dynamics of proteins in solution, these NMR experiments still have several limitations. Each experiment is limited to a narrow time regime and usually requires that data be collected at multiple fields. Further it is still impossible to study states with lifetimes exceeding a few hundred milliseconds. We are developing new methods to address these problems.

Figure 1 Exchange between the different conformational states of T4 Lysozyme can be studied using novel NMR techniques [1, 4, 5]. A) Crystal structure of the major exposed form and NMR derived structure of the minor buried form that has a lifetime of ~1ms [4]. B) NMR spectra of the major form showing only the major state peaks. C) CPMG relaxation dispersion curves recorded from spectra like the one in B were analysed to obtain the minor state structure and obtain the kinetics of the exchange (D). (Figure adapted from [5])

References

[1] Structures of invisible excited protein states by relaxation dispersion NMR spectroscopy. Proc. Natl. Acad. Sci. USA 105, 11766-71.
[2] Increasing the exchange time-scale that can be probed by CPMG relaxation dispersion NMR J Phys Chem B. 115,14891-900.
[3] Studying “Invisible” Excited Protein States in Slow Exchange with a Major State Conformation. J Am Chem Soc 134, 8148-61.
[4] Solution structure of a minor and transiently formed state of a T4 lysozyme mutant. Nature. 477, 111-114.
[5] Atomistic Picture of Conformational Exchange in a T4 Lysozyme Cavity Mutant: An Experiment-Guided Molecular Dynamics Study. Chem Sci. DOI: 10.1039/c5sc03886c in press.