Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by 19F NMR and Cryo-EM.

Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.

Mutations in the X-linked intellectual disability gene, zDHHC9, alter autopalmitoylation activity by distinct mechanisms.

Early onset intellectual disabilities result in significant societal and economic costs and affect 1-3% of the population. The underlying genetic determinants are beginning to emerge and are interpreted in the context of years of work characterizing postsynaptic receptor and signaling functions of learning and memory. DNA sequence analysis of intellectual disability patients has revealed greater than 80 loci on the X-chromosome that are potentially linked to disease. One of the loci is zDHHC9, a gene encoding a Ras protein acyltransferase. Protein palmitoylation is a reversible modification that controls the subcellular localization and distribution of membrane receptors, scaffolds, and signaling proteins required for neuronal plasticity. Palmitoylation occurs in two steps. In the first step, autopalmitoylation, an enzyme-palmitoyl intermediate is formed. During the second step, the palmitoyl moiety is transferred to a protein substrate, or if no substrate is available, hydrolysis of the thioester linkage produces the enzyme and free palmitate. In this study, we demonstrate that two naturally occurring variants of zDHHC9, encoding R148W and P150S, affect the autopalmitoylation step of the reaction by lowering the steady state amount of the palmitoyl-zDHHC9 intermediate.

The crowd you're in with: effects of different types of crowding agents on protein aggregation.

The intracellular environment contains high concentrations of macromolecules occupying up to 30% of the total cellular volume. Presence of these macromolecules decreases the effective volume available for the proteins in the cell and thus increases the effective protein concentrations and stabilizes the compact protein conformations. Macromolecular crowding created by various macromolecules such as proteins, nucleic acids, and carbohydrates has been shown to have a significant effect on a variety of cellular processes including protein aggregation. Most studies of macromolecular crowding have used neutral, flexible polysaccharides that function primarily via excluded volume effect as model crowding agents. Here we have examined the effects of more rigid polysaccharides on protein structure and aggregation. Our results indicate that rigid and flexible polysaccharides influence protein aggregation via different mechanisms and suggest that, in addition to excluded volume effect, changes in solution viscosity and non-specific protein-polymer interactions influence the structure and dynamics of proteins in crowded environments.

Evolutionary analysis reveals the origin of sodium coupling in glutamate transporters.

Secondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to changing environments and needs. The bases of such diversification, and thus principles of ion coupling, are unexplored. Employing phylogenetics and ancestral protein reconstruction, we investigated sodium-coupled transport in prokaryotic glutamate transporters, a mechanism ubiquitous across life domains and critical to neurotransmitter recycling in humans. We found that the evolutionary transition from sodium-dependent to independent substrate binding to the transporter preceded changes in the coupling mechanism. Structural and functional experiments suggest that the transition entailed allosteric mutations, making sodium binding dispensable without affecting ion-binding sites. Allosteric tuning of transporters' energy landscapes might be a widespread route of their functional diversification.

The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding.

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following substrate binding. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. By contrast, an equilibrated structure does not show such classes. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties.

Physicochemical sequence characteristics that influence S-palmitoylation propensity.

Over the past 30 years, several hundred eukaryotic proteins spanning from yeast to man have been shown to be S-palmitoylated. This post-translational modification involves the reversible addition of a 16-carbon saturated fatty acyl chain onto the cysteine residue of a protein where it regulates protein membrane association and distribution, conformation, and stability. However, the large-scale proteome-wide discovery of new palmitoylated proteins has been hindered by the difficulty of identifying a palmitoylation consensus sequence. Using a bioinformatics approach, we show that the enrichment of hydrophobic and basic residues, the cellular context of the protein, and the structural features of the residues surrounding the palmitoylated cysteine all influence the likelihood of palmitoylation. We developed a new palmitoylation predictor that incorporates these identified features, and this predictor achieves a Matthews Correlation Coefficient of .74 using 10-fold cross validation, and significantly outperforms existing predictors on unbiased testing sets. This demonstrates that palmitoylation sites can be predicted with accuracy by taking into account not only physiochemical properties of the modified cysteine and its surrounding residues, but also structural parameters and the subcellular localization of the modified cysteine. This will allow for improved predictions of palmitoylated residues in uncharacterized proteins. A web-based version of this predictor is currently under development.

Quarterly intrinsic disorder digest (January-February-March, 2014).

This is the 5th issue of the Digested Disorder series that represents a reader's digest of the scientific literature on intrinsically disordered proteins. We continue to use only 2 criteria for inclusion of a paper to this digest: The publication date (a paper should be published within the covered time frame) and the topic (a paper should be dedicated to any aspect of protein intrinsic disorder). The current digest issue covers papers published during the first quarter of 2014; i.e., during the period of January, February, and March of 2014. Similar to previous issues, the papers are grouped hierarchically by topics they cover, and for each of the included papers a short description is given on its major findings.

Functional correlations of respiratory syncytial virus proteins to intrinsic disorder.

Protein intrinsic disorder is an important characteristic demonstrated by the absence of higher order structure, and is commonly detected in multifunctional proteins encoded by RNA viruses. Intrinsically disordered regions (IDRs) of proteins exhibit high flexibility and solvent accessibility, which permit several distinct protein functions, including but not limited to binding of multiple partners and accessibility for post-translational modifications. IDR-containing viral proteins can therefore execute various functional roles to enable productive viral replication. Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. In this study, we performed a comprehensive evaluation of predicted intrinsic disorder of the RSV proteome to better understand the functional role of RSV protein IDRs. We included 27 RSV strains to sample major RSV subtypes and genotypes, as well as geographic and temporal isolate differences. Several types of disorder predictions were applied to the RSV proteome, including per-residue (PONDR®-FIT and PONDR® VL-XT), binary (CH, CDF, CH-CDF), and disorder-based interactions (ANCHOR and MoRFpred). We classified RSV IDRs by size, frequency and function. Finally, we determined the functional implications of RSV IDRs by mapping predicted IDRs to known functional domains of each protein. Identification of RSV IDRs within functional domains improves our understanding of RSV pathogenesis in addition to providing potential therapeutic targets. Furthermore, this approach can be applied to other NNS viruses that encode essential multifunctional proteins for the elucidation of viral protein regions that can be manipulated for attenuation of viral replication.

Digested disorder, Quarterly intrinsic disorder digest (October-November-December, 2013).

This is the 4th issue of the Digested Disorder series that represents reader's digest of the scientific literature on intrinsically disordered proteins. The only 2 criteria for inclusion in this digest are the publication date (a paper should be published within the covered time frame) and topic (a paper should be dedicated to any aspect of protein intrinsic disorder). The current digest issue covers papers published during the fourth quarter of 2013; i.e. during the period of October, November, and December of 2013. Similar to previous issues, the papers are grouped hierarchically by topics they cover, and for each of the included paper a short description is given on its major findings.

Digested disorder: Quarterly intrinsic disorder digest (July-August-September, 2013).

The current literature on intrinsically disordered proteins grows fast. To keep interested readers up to speed with this literature, we continue a "Digested Disorder" project and represent a new issue of reader's digest of the research papers and reviews on intrinsically disordered proteins. The only 2 criteria for inclusion in this digest are the publication date (a paper should be published within the covered time frame) and topic (a paper should be dedicated to any aspect of protein intrinsic disorder). The current digest issue covers papers published during the third quarter of 2013; i.e., during the period of June, July, and September of 2013. Similar to previous issues, the papers are grouped hierarchically by topics they cover, and for each of the included paper a short description is given on its major findings.

A putative role of the Sup35p C-terminal domain in the cytoskeleton organization during yeast mitosis.

Sup35 protein (Sup35p), or eukaryotic peptide chain release factor GTP binding subunit (eRF3), is a well-known yeast prion responsible for the characteristic [PSI(+)] trait. N- and M-domains of this protein have been the foci of intensive research due to their importance for the prion formation. Sup35p C-terminal domain (Sup35pC) is essential for translation termination and cell viability. Deletion of Sup35pC was shown to lead to malformation of cells during mitosis. In this study we confirm that Sup35pC domain possesses high sequence and structural similarity to the eukaryotic translation elongation factor 1-α (eEF1A) from yeast and show that its sequence is conserved across different species including human. Because cell malformation during mitosis could be due to the deregulation of cytoskeleton formation, and since a Sup35 paralog eEF1A is known to act as an actin modulating protein, we focused on establishing of the relationships between the Sup35pC and modulation of the cytoskeleton formation. We found 104 co-partners between Sup35pC and EF1A of S. cerevisiae, and 18 partners of human ERF3A. Based on the analysis of known and modeled structures of some effectors and partners we found possible protein-protein interactions. Based on our study, we propose that Sup35pC may serve as actin modulator during mitosis.

Digested disorder: Quarterly intrinsic disorder digest (April-May-June, 2013).

The current literature on intrinsically disordered proteins is overwhelming. To keep interested readers up to speed with this literature, we continue a "Digested Disorder" project and represent a series of reader's digest type articles objectively representing the research papers and reviews on intrinsically disordered proteins. The only 2 criteria for inclusion in this digest are the publication date (a paper should be published within the covered time frame) and topic (a paper should be dedicated to any aspect of protein intrinsic disorder). The current digest issue covers papers published during the period of April, May, and June of 2013. The papers are grouped hierarchically by topics they cover, and for each of the included paper a short description is given on its major findings.