Research Description

NMR of Supra-Molecular Machines; Characterizing Low Populated States of Proteins

Research in the Kay laboratory spans a range of disciplines from spectroscopy and biophysics through to biochemistry. Two major areas of interest include:

NMR of Supra-Molecular Machines

The machinery used by the cell to perform essential  biological processes is made up of large molecular assemblies and insight into their functionally important motions and transient interactions have been very difficult to obtain. We develop new NMR techniques that preserves the NMR signal in a manner such that it is much less affected by the devastating sensitivity losses that normally plague NMR applications to large systems. Using this methodology it has become possible to study systems in quantitative detail with molecular weights as large as one million Daltons. This methodology has been applied to the proteasome, where we have elucidated how dynamics are able to regulate the entry of targets for degradation and the interactions between substrate and the proteasome. Our work explains properties of the proteasome that cannot be understood on the basis of static three-dimensional structures and opens up the possibility of using allosteric drugs to modulate proteasome function.

 

Characterizing Low Populated States of Proteins

Starting from the seminal work of Max Perutz and others in the 1950s a main goal of structural biology has been to elucidate three-dimensional images of protein molecules. These pictures are typically obtained under conditions where the most stable state of the protein is studied, ignoring higher energy conformations. Yet it is becoming increasingly appreciated that proteins are not static entities and that their biological function is dictated in many cases by excursions between conformations with different energies. We develop NMR spin relaxation experiments that allow one to ‘see the invisible’ by measuring chemical shifts of and bond vector orientations in high energy conformational states of proteins, even when such states do not produce observable spectra. These NMR measurements can in turn be used with  computational approaches to produce atomic resolution models and insight into biological function.

Publications

View all publications on PubMed

Measurement of active site ionization equilibria in the 670 kDa proteasome core particle using methyl-TROSY NMR
Velyvis A, Kay LE.
J Am Chem Soc. 2013 Jun 26;135(25):9259-62. doi: 10.1021/ja403091c  Read

Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction
Rosenzweig R, Moradi S, Zarrine-Afsar A, Glover JR, Kay LE.
Science. 2013 Mar 1;339(6123):1080-3. doi: 10.1126/science.1233066  Read

Proteasome allostery as a population shift between interchanging conformers
Ruschak AM, Kay LE.
Proc Natl Acad Sci U S A. 2012 Nov 20;109(47):19268-73. doi: 10.1073/pnas.1212036109  Read

Folding of the four-helix bundle FF domain from a compact on-pathway intermediate state is governed predominantly by water motion
Sekhar A, Vallurupalli P, Kay LE.
Proc Natl Acad Sci U S A. 2012 Nov 20;109(47):19268-73. doi: 10.1073/pnas.1212036109  Read

Structure of an intermediate state in protein folding and aggregation
Neudecker P, Robustelli P, Cavalli A, Walsh P, Lundström P, Zarrine-Afsar A, Sharpe S, Vendruscolo M, Kay LE.
Science. 2012 Apr 20;336(6079):362-6. doi: 10.1126/science.1214203  Read

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant
Bouvignies G, Vallurupalli P, Hansen DF, Correia BE, Lange O, Bah A, Vernon RM, Dahlquist FW, Baker D, Kay LE.
Nature. 2011 Aug 21;477(7362):111-4. doi: 10.1038/nature10349  Read