Osmolyte-induced folding of an intrinsically disordered protein: folding mechanism in the absence of ligand.

Understanding the interconversion between thermodynamically distinguishable states present in a protein folding pathway provides not only the kinetics and energetics of protein folding but also insights into the functional roles of these states in biological systems. The protein component of the bacterial RNase P holoenzyme from Bacillus subtilis (P protein) was previously shown to be unfolded in the absence of its cognate RNA or other anionic ligands. P protein was used in this study as a model system to explore general features of intrinsically disordered protein (IDP) folding mechanisms. The use of trimethylamine N-oxide (TMAO), an osmolyte that stabilizes the unliganded folded form of the protein, enabled us to study the folding process of P protein in the absence of ligand. Transient stopped-flow kinetic traces at various final TMAO concentrations exhibited multiphasic kinetics. Equilibrium "cotitration" experiments were performed using both TMAO and urea during the titration to produce a urea-TMAO titration surface of P protein. Both kinetic and equilibrium studies show evidence of a previously undetected intermediate state in the P protein folding process. The intermediate state is significantly populated, and the folding rate constants are relatively slow compared to those of intrinsically folded proteins similar in size and topology. The experiments and analysis described serve as a useful example for mechanistic folding studies of other IDPs.

Investigating the Structure of FtsZ to Understand its Functional Role in Bacterial Cell Division

FtsZ, a bacterial tubulin homologue, is a cytoskeleton protein that plays key roles in cytokinesis of almost all prokaryotes. FtsZ assembles into protofilaments (pfs), one subunit thick, and these pfs assemble further to form a “Z ring” at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane, and also serves as a scaffold to recruit cell-wall remodeling proteins for complete cell division in vivo. FtsZ can be subdivided into 3 main functional regions: globular domain, C terminal (Ct) linker, and Ct peptide. The globular domain binds GTP to assembles the pfs. The extreme Ct peptide binds membrane proteins to allow cytoplasmic FtsZ to function at the inner membrane. The Ct linker connects the globular domain and Ct peptide. In the present studies, we used genetic and structural approaches to investigate the function of Escherichia coli (E. coli) FtsZ. We sought to examine three questions: (1) Are lateral bonds between pfs essential for the Z ring? (2) Can we improve direct visualization of FtsZ in vivo by engineering an FtsZ-FP fusion that can function as the sole source of FtsZ for cell division? (3) Is the divergent Ct linker of FtsZ an intrinsically disordered peptide (IDP)?

One model of the Z ring proposes that pfs associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of E. coli FtsZ by inserting either small peptides or whole FPs. Of the four lateral surfaces on FtsZ pfs, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174 located on the left and right surfaces, completely blocked function, and were identified as possible sites for essential lateral interactions. Another goal was to find a location within FtsZ that supported fusion of FP reporter proteins, while allowing the FtsZ-FP to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by super-resolution techniques.

The Ct linker is the most divergent region of FtsZ in both sequence and length. In E. coli FtsZ the Ct linker is 50 amino acids (aa), but for other FtsZ it can be as short as 37 aa or as long as 250 aa. The Ct linker has been hypothesized to be an IDP. In the present study, circular dichroism confirmed that isolated Ct linkers of E. coli (50 aa) and C. crescentus (175 aa) are IDPs. Limited trypsin proteolysis followed by mass spectrometry (LC-MS/MS) confirmed Ct linkers of E. coli (50 aa) and B. subtilis (47 aa) as IDPs even when still attached to the globular domain. In addition, we made chimeras, swapping the E. coli Ct linker for other peptides and proteins. Most chimeras allowed for normal cell division in E. coli, suggesting that IDPs with a length of 43 to 95 aa are tolerated, sequence has little importance, and electrostatic charge is unimportant. Several chimeras were purified to confirm the effect they had on pf assembly. We concluded that the Ct linker functions as a flexible tether allowing for force to be transferred from the FtsZ pf to the membrane to constrict the septum for division.

Optimized, unequal pulse spacing in multiple echo sequences improves refocusing in magnetic resonance.

A recent quantum computing paper (G. S. Uhrig, Phys. Rev. Lett. 98, 100504 (2007)) analytically derived optimal pulse spacings for a multiple spin echo sequence designed to remove decoherence in a two-level system coupled to a bath. The spacings in what has been called a "Uhrig dynamic decoupling (UDD) sequence" differ dramatically from the conventional, equal pulse spacing of a Carr-Purcell-Meiboom-Gill (CPMG) multiple spin echo sequence. The UDD sequence was derived for a model that is unrelated to magnetic resonance, but was recently shown theoretically to be more general. Here we show that the UDD sequence has theoretical advantages for magnetic resonance imaging of structured materials such as tissue, where diffusion in compartmentalized and microstructured environments leads to fluctuating fields on a range of different time scales. We also show experimentally, both in excised tissue and in a live mouse tumor model, that optimal UDD sequences produce different T(2)-weighted contrast than do CPMG sequences with the same number of pulses and total delay, with substantial enhancements in most regions. This permits improved characterization of low-frequency spectral density functions in a wide range of applications.

HDAC6 and Ubp-M BUZ domains recognize specific C-terminal sequences of proteins.

The BUZ/Znf-UBP domain is a protein module found in the cytoplasmic deacetylase HDAC6, E3 ubiquitin ligase BRAP2/IMP, and a subfamily of ubiquitin-specific proteases. Although several BUZ domains have been shown to bind ubiquitin with high affinity by recognizing its C-terminal sequence (RLRGG-COOH), it is currently unknown whether the interaction is sequence-specific or whether the BUZ domains are capable of binding to proteins other than ubiquitin. In this work, the BUZ domains of HDAC6 and Ubp-M were subjected to screening against a one-bead-one-compound (OBOC) peptide library that exhibited random peptide sequences with free C-termini. Sequence analysis of the selected binding peptides as well as alanine scanning studies revealed that the BUZ domains require a C-terminal Gly-Gly motif for binding. At the more N-terminal positions, the two BUZ domains have distinct sequence specificities, allowing them to bind to different peptides and/or proteins. A database search of the human proteome on the basis of the BUZ domain specificities identified 11 and 24 potential partner proteins for Ubp-M and HDAC6 BUZ domains, respectively. Peptides corresponding to the C-terminal sequences of four of the predicted binding partners (FBXO11, histone H4, PTOV1, and FAT10) were synthesized and tested for binding to the BUZ domains by fluorescence polarization. All four peptides bound to the HDAC6 BUZ domain with low micromolar K(D) values and less tightly to the Ubp-M BUZ domain. Finally, in vitro pull-down assays showed that the Ubp-M BUZ domain was capable of binding to the histone H3-histone H4 tetramer protein complex. Our results suggest that BUZ domains are sequence-specific protein-binding modules, with each BUZ domain potentially binding to a different subset of proteins.

Evolution of networks and sequences in eukaryotic cell cycle control.

The molecular networks regulating the G1-S transition in budding yeast and mammals are strikingly similar in network structure. However, many of the individual proteins performing similar network roles appear to have unrelated amino acid sequences, suggesting either extremely rapid sequence evolution, or true polyphyly of proteins carrying out identical network roles. A yeast/mammal comparison suggests that network topology, and its associated dynamic properties, rather than regulatory proteins themselves may be the most important elements conserved through evolution. However, recent deep phylogenetic studies show that fungal and animal lineages are relatively closely related in the opisthokont branch of eukaryotes. The presence in plants of cell cycle regulators such as Rb, E2F and cyclins A and D, that appear lost in yeast, suggests cell cycle control in the last common ancestor of the eukaryotes was implemented with this set of regulatory proteins. Forward genetics in non-opisthokonts, such as plants or their green algal relatives, will provide direct information on cell cycle control in these organisms, and may elucidate the potentially more complex cell cycle control network of the last common eukaryotic ancestor.