Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy.

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side-chain dynamics of the α-helical sensory rhodopsin II and the ß-barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl-bearing side-chain motion that is largely independent of membrane mimetic. The methyl-bearing side chains of both proteins are, on average, more dynamic in the ps-ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane-protein functions, including ligand binding, allostery, and signaling.

Improving yields of deuterated, methyl labeled protein by growing in H2O.

Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the 15N-1H TROSY and 13C-1H3 methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in D2O leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins. Expression of protein during growth in H2O, with the addition of 2H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in H2O M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in D2O M9 for a variety of protein targets while still achieving 75-80% deuteration. Because the procedure does not require bulk D2O or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific 13CH3 labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of 2H/15N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.

Comprehensive pharmacological profiling of neurofibromatosis cell lines.

Patients with Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are predisposed to tumors of the nervous system. NF1 patients predominantly develop neurofibromas, and Malignant Peripheral Nerve Sheath Tumors (MPNST) while NF2 patients develop schwannomas and meningiomas. Here we quantified the drug sensitivities of NF1 and NF2 tumor cell lines in a high throughput platform. The platform contained a comprehensive collection of inhibitors of MEK, RAF, RAS, farnesyl transferase, PAK and ERK, representative drugs against many other cancer pathways including Wnt, Hedgehog, p53, EGF, HDAC, as well as classical cytotoxic agents recommended for treating MPNST, such as doxorubicin and etoposide. We profiled seven NF1-associated MPNST cell lines (ST88-14, ST88-3, 90-8, sNF02.2, T265, S462TY, SNF96.2), one sporadic MPNST cell line (STS26), one schwannoma from a NF2 patient (HEI193), one NF2-deficient malignant meningioma (KT21-MG-Luc5D), one mouse NF2 schwannoma (SC4) and one sporadic rat schwannoma (RT4-67 or RT4). NF1 cells were primarily distinguished from NF2 cells and the sporadic MPNST cell line by their sensitivity to MEK and ERK inhibitors, and to a smaller extent their sensitivity to BH3 mimetics and farnesyl transferase inhibitors. The platform was highly successful in predicting the effects of clinical trials for Neurofibromas.