Prof. Joel L. Sussman

Studies on Intrinsically Unstructured Proteins.

A key protein recognition challenge is faced by the Sussman group, which for many years has focused on key proteins in the nervous system including acetylcholinesterase (AChE) (Sussman et al Silman, Science. 1991;253:872–879) and the cholinesterase-like adhesion molecules (CLAMs) (Paz et al & Sussman & Silman Biophys J. 2008;95:1928–1944). The CLAMs share a common extra-cytoplasmic 3D structure homologous to that of AChE, but are devoid of its catalytic triad, and thus are not enzymes. Rather they are adhesion proteins involved in the earliest stages of the development of the nervous system. Their cytoplasmic domain (tail) consists of ~250 amino acids that are intrinsically disordered (ID). IDs represent about 1/3 of eukaryotic proteins ( Dunker  et al & Sussman, Curr Opin Struct Biol. 2008;18:756–764).

CLAMs will be used as a biologically interesting model system to investigate the nature of ID proteins, with the overall goal of placing the phenomenon on firmer theoretical and experimental grounds, and addressing the following key questions: Are there distinct classes of intrinsically disordered proteins (IDPs)/intrinsically disordered domains (IDDs), or are they a continuum? Can IDPs be forced to fold in the presence of osmolyte co-solvents? Is ID abolished when the protein enters the crowded cellular environment, at least in some instances? And, if so, does this occur at a particular developmental stage, in specific cellular compartments, and/or upon interaction with one or more binding partners?

Working closely with Frydman, methods are being developed to examine these proteins in living cells via NMR. A number of difficult hurdles will have to be overcome for the success of this project, including:

  1. signal overlap and
  2. the low sensitivity of in-cell NMR.

In order to achieve adequate signal/noise (S/N) ratios, in-cell NMR methods currently require over-expression of the target protein to levels of 2-5% of the total soluble protein. The current detection limit for 15N-1H HSQC based in-cell NMR experiments is ~200 μM, while the corresponding S/N ratio for methyl group 13C-1H HSQC experiments is ~70 μM. By use of the 950 MHz spectrometer requested as part of this I-CORE, together with a state-of-the-art CryoProbe, it will be possible to obtain significantly higher sensitivity, i.e. a S/N increase of 10-15-fold compared to the values obtained using a 600 MHz spectrometer with a room-temperature probe, thus reducing both the protein concentration and the experimental time needed to collect the NMR data. In this latter respect, we trust that the application of new NMR experimental methods being developed at WIS will contribute both to enhancement of the S/N ratio and to resolution.

Finally, in collaborations with Minsky, super-resolution light microscopy (particularly SIM) and correlative light/electron microscopy will be used to complement these atomic resolution methods to follow the locations of the CLAMs in living cells.