Remote homology searches identify bacterial homologues of eukaryotic lipid transfer proteins, including Chorein-N domains in TamB and AsmA and Mdm31p

BACKGROUND: All cells rely on lipids for key functions. Lipid transfer proteins allow lipids to exit the hydrophobic environment of bilayers, and cross aqueous spaces. One lipid transfer domain fold present in almost all eukaryotes is the TUbular LIPid binding (TULIP) domain. Three TULIP families have been identified in bacteria (P47, OrfX2 and YceB), but their homology to eukaryotic proteins is too low to specify a common origin. Another recently described eukaryotic lipid transfer domain in VPS13 and ATG2 is Chorein-N, which has no known bacterial homologues. There has been no systematic search for bacterial TULIPs or Chorein-N domains. RESULTS: Remote homology predictions for bacterial TULIP domains using HHsearch identified four new TULIP domains in three bacterial families. DUF4403 is a full length pseudo-dimeric TULIP with a 6 strand β-meander dimer interface like eukaryotic TULIPs. A similar sheet is also present in YceB, suggesting it homo-dimerizes. TULIP domains were also found in DUF2140 and in the C-terminus DUF2993. Remote homology predictions for bacterial Chorein-N domains identified strong hits in the N-termini of AsmA and TamB in diderm bacteria, which are related to Mdm31p in eukaryotic mitochondria. The N-terminus of DUF2993 has a Chorein-N domain adjacent to its TULIP domain. CONCLUSIONS: TULIP lipid transfer domains are widespread in bacteria. Chorein-N domains are also found in bacteria, at the N-terminus of multiple proteins in the intermembrane space of diderms (AsmA, TamB and their relatives) and in Mdm31p, a protein that is likely to have evolved from an AsmA/TamB-like protein in the endosymbiotic mitochondrial ancestor. This indicates that both TULIP and Chorein-N lipid transfer domains may have originated in bacteria

Caught green-handed : methods for in vivo detection and visualization of protease activity

Proteases are enzymes that cleave peptide bonds of other proteins. Their omnipresence and diverse activities make them important players in protein homeostasis and turnover of the total cell proteome as well as in signal transduction in plant stress response and development. To fully understand protease function, it is of paramount importance to assess when and where a specific protease is active. Here, we review the existing methods to detect in vivo protease activity by means of imaging chemical activity-based probes and genetically encoded sensors. We focus on the diverse fluorescent and luminescent sensors at the researcher's disposal and evaluate the potential of imaging techniques to deliver in vivo spatiotemporal detail of protease activity. We predict that in the coming years, revised techniques will help to elucidate plant protease activity, functions and hence expand the current status of the field

Optoelectrical dynamics of ion channels and subcellular calcium nanodomains.

Countless investigations have used highly invasive electrophysiology or non-dynamic biochemical approaches to study synapses and networks. Optogenetic approaches, combined with imaging techniques, are revolutionary tools to excite specific live cells and detect the activity of signaling molecules in populations of neurons. However, refinement of current optical methods is needed, due to the lack of molecular or spatial specificity. This refinement is particularly important for imaging Ca2+ in neurons, since Ca2+ sig- nals exert their highly specific functions in well-defined cellular subcompartments. Coupling of Ca2+ signaling to membrane voltage occurs in Ca2+ nanodomains where Ca2+ influx through voltage-gated Ca2+ channels is located within 10-50 nm of BK K+ channels. Upon Ca2+ entry, BK open in response to additive effects of Ca2+ and volt- age to limit neuronal excitability. Nevertheless, much remains to be known about this process, since currently no sensors located specifically to these regions are avail- able. Developing probes that provide bright readouts in vivo combined with ex- tremely fast and high spatial resolution imaging systems is crucial to progress to- wards our knowledge about neuronal networking

Use of Peptide Libraries for Identification and Optimization of Novel Antimicrobial Peptides.

The increasing rates of resistance among bacteria and to a lesser extent fungi have resulted in an urgent need to find new molecules that hold therapeutic promise against multidrug-resistant strains. Antimicrobial peptides have proven very effective against a variety of multidrug-resistant bacteria. Additionally, the low levels of resistance reported towards these molecules are an attractive feature for antimicrobial drug development. Here we summarise information on diverse peptide libraries used to discover or to optimize antimicrobial peptides. Chemical synthesized peptide libraries, for example split and mix method, tea bag method, multi-pin method and cellulose spot method are discussed. In addition biological peptide library screening methods are summarized, like phage display, bacterial display, mRNA-display and ribosomal display. A few examples are given for small peptide libraries, which almost exclusively follow a rational design of peptides of interest rather than a combinatorial approach

A unified evolutionary origin for the ubiquitous protein transporters SecY and YidC.

BACKGROUND: Protein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known. RESULTS: The features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY's halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC's sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains. CONCLUSIONS: SecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells

Understanding the replication biology of Providence virus: elucidating the function of non-structural proteins

Tetraviruses are non-enveloped, small insect RNA viruses with a single stranded positive RNA genome that is either monopartite or bipartite. Providence virus (PrV) is the only member of the three tetravirus families with a viral replicase similar to the replicases of tombusviruses and umbraviruses. The principle aim of this thesis was to study PrV replication, focusing on subcellular localization and potential interactions between PrV replication proteins. The first objective of this study was to generate an anti-p104 antibody that does not cross-react with p40. Expression of the C-terminal portion of p104 in E. coli resulted in no detectable protein. Further expression in an insect cell based expression system resulted in the production of an insoluble protein. Attempts to improve protein solubility with a range of solubilization treatments were unsuccessful. Bioinformatic analysis was used to detect an antigenic region at the C-terminus of p104 and the peptide was used to raise anti-p104 antibodies. These antibodies did not detect native protein by western blot detection however they were used for immunoprecipitation. The establishment of the subcellular localization of PrV required two approaches; immunofluorescence in persistently infected Helicoverpa zea MG8 cells using antip40 and anti-dsRNA antibodies and the expression of EGFP-replicase fusion protein in Spodoptera frugiperda Sf9 cells. Replication of PrV was found to take place in cytosolic punctate structures. Co-immunoprecipitation experiments revealed that p40 self-interacts and interacts with p104. Bioinformatic analysis of PrV p104 suggests that the RdRp is similar to viral RdRps of the carmo-like supergroup II. Potential RNA binding regions are present within p104. A potential p40 interaction domain that shares hydrophilic and surface exposed properties with the TBSV p33 interaction domain is present. A putative arginine-rich region and disordered C-terminal region is present in p130. In conclusion, PrV p104 is the viral replicase. The resemblance of the expression strategy and putative functional domains with tombusviruses and umbraviruses suggest that PrV replication is related to the replication system of the tombusviruses and umbraviruses. This has led to propose that tetravirus replication strategies are diverse and raises questions on the origin and evolution of PrV.Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 201

Towards Understanding How Membrane Proteins Approach And Fold Into Membranes

Membrane proteins must fold into phospholipid bilayers to function. Some membrane proteins are cotranslationally inserted into the membrane, while others rely on chaperone networks to solubilize and traffic unfolded membrane proteins to the membrane. In my thesis work I have studied both facets of membrane protein folding: chaperone interactions and insertion into the membrane. The first part of my thesis investigates the intrinsic conformational properties and unfolded outer membrane protein (uOMP) binding of the main chaperone in OMP biogenesis pathway in E. coli: SurA. We found that SurA is monomeric and exists in three major conformations in solution. Next, we mapped the uOMP binding site on SurA, finding that the least intrinsically populated conformation is the chaperone-active state. Using a plethora of experimental data as restraints, we constructed a model of the SurA-uOMP complex in which the uOMP is greatly expanded by SurA. In the second part of my thesis I examined the effect of the local environment imposed by both the protein and bilayer on individual side chain transfer free energies. I found that the transfer free energies for most side chains are only slightly altered by changes in neighboring side chains and packing. By relating the local composition of the membrane to nonpolar side chain transfer free energies, I discovered a linear correlation between the nonpolar solvation parameter and local concentration of water in the bilayer. Together these studies highlight the structural and thermodynamic parameters that drive efficient membrane protein biogenesis and folding

Modulating Calcium Signaling by Protein Design and Analysis of Calcium Binding Proteins

Transient change of cytosolic calcium level leads to physiological actions, which are modulated by the intracellular calcium stores, and gated by membrane calcium channels/pumps. To closely monitor calcium dynamics there is a pressing need to develop calcium sensors that are targeted to high calcium environment such as the ER/SR with relatively low binding affinity and fast kinetic properties to complement the current calcium indicator toolkits. In this dissertation, the development of fast red florescent calcium binding protein using the protein design is reported. The results show the calcium dependent fluorescence increase of mCherry mutant MCD1 (RapidER) and MCD15 (RapidER’) is able to monitor the ER calcium release in several cell lines responding to perturbations of extracellular calcium signaling. The specific targeting to the ER membrane was achieved by fusing the ryanodine receptor 1 transmembrane domains for the spatio-temporal calcium imaging. To understand the underlying mechanism of calcium binding induced fluorescence increase in the designed calcium sensor CatchER, the fluorescence lifetime of CatchER was determined in calcium free and bound forms using time resolved florescence spectroscopy. The results suggest that calcium binding inhibits the geminate quenching, resulting in a longer lifetime when the anionic form is indirectly excited at 395 nm. It is believed that such unique calcium-induced lifetime change can be applied to monitor calcium signaling in cell imaging. NMR spectroscopy was used to investigate the protein-protein/ligand interaction in this dissertation. The residual dipolar coupling and T1, T2, NOE dynamic study were carried out to understand the binding mode of CaM and the N-terminal intracellular loop of connexin 43. The results show that both N and C terminal domains of Ca2+-CaM contact with the peptide, leading to a partially unwound and bending central helix of CaM. The ligand binding induced conformational change was demonstrated by selectively labeled proteins including extracellular domain of calcium sensing receptor and the bacterial membrane protein SecA fragments C34 and N68

Evolutionary engineering of green fluorescent protein calcium biosensors

Neurobiology continues to be one of the great frontiers in biological sciences. The number of neurons in the brain, and the complex neuronal circuits they constitute, will keep scientists trying to decipher them challenged for years to come. In the last decade, the use of genetically encoded calcium indicators (GECIs) to monitor and visualize neuronal activity has greatly advanced. Calcium imaging using GECIs has become a principal modality to elucidate neuronal coding and signaling processes. GECIs provide clear advantages over synthetic calcium dyes by enabling long-term expression and chronic imaging in targeted neurons in vivo. Whilst most improvements of GECIs have been primarily focusing on faster kinetics, calcium sensitivity, brightness and signal strength; less attention has been on GECIs’ likely impact on cellular environments via calcium buffering. Studies have shown that long-term expression of GECIs at high intracellular concentrations can lead to pathological changes and reduced responsiveness in cells. The objective of this dissertation was to design a new family of GECIs suitable for long-term monitoring of neuronal calcium activity. In contrast to previous optimization strategies, here a new species of calcium binding protein, troponin C from Opsanus tau, was used as a basis for the development of a minimal calcium-binding domain. The minimal domain was fused to brighter fluorescent proteins to generate novel GECIs with improved properties. Consequently, the novel GECIs were optimized through iterative rounds of directed molecular evolution and screening, resulting in the Twitch-family of GECIs. In Chapter 2, we describe the structure-function relationships of a previously published FRET-based calcium indicator, the TN-XXL. The structure-function relationship in FRET- based GECIs is largely uncharacterized due to the artificial and multi-modular composition. By utilizing a combination of protein engineering, spectroscopic and biophysical analyses, we show that two of the four calcium binding sites dominate the FRET output. Furthermore, we found that local conformational changes of these sites match the kinetics of FRET change. We show that TN-XXL changes from a flexible elongated structure to a rigid globular shape upon binding calcium. The insights gained from this work formed the basis for the engineering of the FRET-based GECIs described in this work. In Chapter 3, a newly developed minimal domain FRET-based GECI, Twitch-1CD, was introduced into auto-antigen-specific and non–auto-antigen-specific CD4+ T cells. We demonstrated for the first time in vivo how a GECI is fully expressed in T cells, and thus allowing for detailed recording and visualization of calcium signaling during T cell antigen- recognition. In Chapter 4, we orchestrated the evolution of the Twitch-family of GECIs, with better signal- to-noise ratios (SNR), greater dynamic range (∆R/R) and calcium kinetics. These indicators underwent rational design and directed molecular evolution, followed by bacterial plate screening and a fluorescent imaging screening assay in hippocampal neurons. The novel GECIs were subsequently applied in a series of studies, emphasizing their improvements to previous FRET-based GECIs