Mapping Protein Conformational Landscapes under Strongly Native Conditions with Hydrogen Exchange Mass Spectrometry.

The thermodynamic stability and kinetic barriers separating protein conformations under native conditions are critical for proper protein function and for understanding dysfunction in diseases of protein conformation. Traditional methods to probe protein unfolding and folding employ denaturants and highly non-native conditions, which may destabilize intermediate species or cause irreversible aggregation, especially at the high protein concentrations typically required. Hydrogen exchange (HX) is ideal for detecting conformational behavior under native conditions without the need for denaturants, but detection by NMR is limited to small highly soluble proteins.

Engineered solubility tag for solution NMR of proteins.

The low solubility of many proteins hinders large scale expression and purification as well as biophysical measurements. Here, we devised a general strategy to solubilize a protein by conjugating it at a solvent-exposed position to a 6 kDa protein that was re-engineered to be highly soluble. We applied this method to the CARD domain of Apoptosis-associated speck-like protein containing a CARD (ASC), which represents one member of a class of proteins that are notoriously prone to aggregation.

Proteasome allostery as a population shift between interchanging conformers.

Protein degradation plays a critical role in cellular homeostasis, in regulating the cell cycle, and in the generation of peptides that are used in the immune response. The 20S proteasome core particle (CP), a barrel-like structure consisting of four heptameric protein rings stacked axially on top of each other, is central to this process. CP function is controlled by activator complexes that bind 75 Å away from sites catalyzing proteolysis, and biochemical data are consistent with an allosteric mechanism by which binding is communicated to distal active sites.

An economical method for production of (2)H, (13)CH3-threonine for solution NMR studies of large protein complexes: application to the 670 kDa proteasome.

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby (13)CH(3) methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-(2)H; β-(2)H;γ-(13)C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics.

Novel proteasome inhibitors to overcome bortezomib resistance.

The proteasome is an intracellular enzyme complex that degrades ubiquitin-tagged proteins and thereby regulates protein levels within the cell. Given this important role in maintaining cellular homeostasis, it is perhaps somewhat surprising that proteasome inhibitors have a therapeutic window. Proteasome inhibitors have demonstrated clinical efficacy in the treatment of multiple myeloma and mantle cell lymphoma and are under evaluation for the treatment of other malignancies.

Site-directed methyl group labeling as an NMR probe of structure and dynamics in supramolecular protein systems: applications to the proteasome and to the ClpP protease.

Methyl groups are powerful reporters of structure, motion, and function in NMR studies of supramolecular protein assemblies. Their utility can be hindered, however, by spectral overlap, difficulties with assignment or lack of probes in biologically important regions of the molecule studied. Here we show that (13)CH(3)-S- labeling of Cys side chains using (13)C-methyl-methanethiosulfonate ((13)C-MMTS) (IUPAC name: methylsulfonylsulfanylmethane) provides a convenient probe of molecular structure and dynamics.

A simple strategy for ¹³C, ¹H labeling at the Ile-γ2 methyl position in highly deuterated proteins.

A straightforward approach for the production of highly deuterated proteins labeled with ¹³C and ¹H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile ¹³C(γ)²,¹H(γ)² HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile ¹³C(δ)¹,¹H(δ)¹ spectra.

The proteasome antechamber maintains substrates in an unfolded state.

Eukaryotes and archaea use a protease called the proteasome that has an integral role in maintaining cellular function through the selective degradation of proteins. Proteolysis occurs in a barrel-shaped 20S core particle, which in Thermoplasma acidophilum is built from four stacked homoheptameric rings of subunits, α and β, arranged α(7)β(7)β(7)α(7) (ref. 5).

Methyl groups as probes of supra-molecular structure, dynamics and function.

The development of new protein labeling strategies, along with optimized experiments that exploit the label, have significantly impacted on the types of biochemical problems that can now be addressed by solution NMR spectroscopy. Here we describe how methyl labeling of key residues in a highly deuterated protein background has facilitated studies of the structure, dynamics and interactions of supra-molecular particles. The methyl-labeling approach is briefly reviewed, followed by a summary of applications to three different molecular machines so as to illustrate the types of questions that can now be addressed.

The role of prefibrillar structures in the assembly of a peptide amyloid.

The self-assembly of proteins into stable, fibrillar aggregates is a general property of polypeptides most notably associated with degenerative diseases termed amyloidoses. These nano- to micrometer scale structures are formed predominantly of beta-sheets that self-assemble by a nucleation-dependent mechanism. The rate-limiting step of assembly involves stabilization of high-energy intermediates in a kinetic step termed nucleation.

Fiber-dependent amyloid formation as catalysis of an existing reaction pathway.

A central component of a number of degenerative diseases is the deposition of protein as amyloid fibers. Self-assembly of amyloid occurs by a nucleation-dependent mechanism that gives rise to a characteristic sigmoidal reaction profile. The abruptness of this transition is a variable characteristic of different proteins with implications to both chemical mechanism and the aggressiveness of disease.

Secondary structure models of the 3' untranslated regions of diverse R2 RNAs.

The RNA structure of the 3' untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure-function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3' UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3' UTR R2 RNA of Forficula auricularia.