Structure-function relationship of viral coat proteins : a site-directed spectroscopic study of M13 coat protein

This thesis describes the results of a spectroscopic study of the major coat protein of bacteriophage M13. During the infection process this protein is incorporated into the cytoplasmic membrane of Escherichia coli host cells. To specifically monitor the local structural changes and changes in the environment of the protein upon membrane insertion, a set of cysteine site-specific mutants of protein was produced for the purpose of ESR spin labeling and fluorescence spectroscopy. These spectroscopic techniques, in combination with CD spectroscopy, are particularly suitable for comparison of protein structural changes in different membrane model systems. The spectroscopic experiments indicate that the very tight structure of the phage particle was disrupted only with strong ionic detergents, such as SDS and CTAB. However the phage structure was not affected by either lipids or nonionic detergents. On the other hand, after a chloroform-induced transformation of the filamentous phage particle into the S- form, the major coat protein was completely solubilized under these conditions. Upon solubilization of the phage particle in sodium cholate at low pH, a protein "structural dimer" appeared to be the most stable aggregate. This structural dimer, in which the protein subunits that are slightly shifted with respect to each other, is proposed to play a key role in the assembly and disassembly of the phage particle in vivo. However, when completely solubilized in the membrane, the major coat protein is stable in a monomer state, and does not have a tendency to aggregate. Site-directed ESR spin labeling was found to be a useful technique to compare the protein structure and topology in micellar and lipid bilayer model systems. To allow a stable association with different membrane model systems, the local structure of the major coat protein changed significantly, but surprisingly the major structural elements, such as the α-helix content, are largely retained. The detailed topology of the major coat protein in lipid bilayers was determined by using Ni 2+quenchers with the spin-labeled mutants. The results show that the part of the major coat protein around amino acid residue Thr36 is situated in the centre of the membrane. Amino acid residues 25 and 46 are located in the lipid head group region at the two water-membrane interfaces, with only a short part of the C-terminus (three to four amino acid residues) extending into the aqueous phase. This transmembrane topology leaves the α-carbons of Lys40, Lys43, and Lys44 in the membrane interior, while the ε-amino groups of the lysine side chains probably interact with the large excess of phosphates in the lipid head groups. Since the major coat protein has to aggregate to form a protective coat around viral DNA, these results indicate that lipids should be removed first at the assembly site, before the major coat protein can interact with DNA to form a new virus coat

A Fresh Look at the Stratigraphy of Northern Australe

The roughly circular collection of mare deposits centered at ~38.9S, 93E is often re- ferred to as Mare Australe. It is located outside of the Procellarum KREEP Terrain. The circular arrangement of Australes mare patches has suggested an ancient, heavily degraded or relaxed impact basin roughly 900 km in diameter. The mare deposits are generally thought to have erupted into smaller post- basin craters. The type, volume, and distribution of mare eruptions potentially resembles the early stages of basin-filling mare events, but which are preserved in Australe and some farside locations. Gravity data suggest that if there was a basin, it is much smaller than originally proposed (now ~600 km) and located in the northern part of Mare Australe, between Humboldt, Milne, and Jenner craters. As a whole, Mare Australe lacks the topography typically associated with a basin; however, northern Australe has a slight topographic depression that roughly corresponds to the basin-like Bouguer gravity signature in the same area. The compositions exposed in Humboldt crater suggest that a preexisting basin might have excavated deeper crustal material. However, the underlying cause of the circularity of Mare Australes deposits, particularly those extending outside of the potential impact basin setting, is not yet understood. Thus, Australe may preserve fundamental information about mare volcanism potentially uncoupled from basin formation and structure. The objectives of this study are to use new high- resolution data (images, gravity, topography, and com- position) to reassess Australes mare deposits, deter- mine the timing and style of volcanism, identify discrete basalt deposits, and to further characterize the evolution of magmatism and subsurface structure in this area. Here, we focus on the northern Australe deposits (between Humboldt, Jenner, and Milne). As originally noted by Whitford-Stark (1979), Humboldt crater and its ejecta make an excellent stratigraphic marker that can be traced across much of the Australe region. The ejecta serves as a stratigraphic constraint for absolute model ages (AMAs) derived from crater size-frequency distributions (CSFDs)

Morphologic Parameters for Successful Lunar Landing Sites

The Moon, with its abundant resources, intriguing science questions, and vast unexplored surface area, is the most attainable and useful near-term target for future human exploration. In recognition of this fact, Presidential Space Policy Directive 1 (PSPD-1) has directed the United States to return to the Moon for long-term exploration and utilization, beginning with the 7th American human lunar landing by 2024 and building to sustainable surface presence by 2028

Viruses: incredible nanomachines. New advances with filamentous phages

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account

Science Enabling Exploration: Using LRO to Prepare for Future Missions

Discoveries from LRO have transformed our understanding of the Moon, but LRO's instruments were originally designed to collect the measurements required to enable future lunar surface exploration. A high lunar exploration priority is the collection of new samples and their return to Earth for comprehensive analysis. The importance of sample return from South Pole-Aitken is well-established [Jolliff et al., this conference], but there are numerous other locations where sample return will yield important advances in planetary science. Using new LRO data, we have defined an achievability envelope based on the physical characteristics of successful lunar landing sites. Those results were then used to define 1km x 1km regions of interest where sample return could be executed, including: the basalt flows in Oceanus Procellarum (22.1N, 53.9W), the Gruithuisen Domes (36.1N, 39.7W), the Dewar cryptomare (2.2S, 166.8E), the Aristarchus pyroclastic deposit (24.8N, 48.5W), the Sulpicius Gallus formation (19.9N, 10.3E), the Sinus Aestuum pyroclastic deposit (5.2N, 9.2W), the Compton-Belkovich volcanic complex (61.5N, 99.9E), the Ina Irregular Mare Patch (18.7N, 5.3E), and the Marius Hills volcanic complex (13.4N, 55.9W). All of these locations represent safe landing sites where sample returns are needed to advance our understanding of the evolution of the lunar interior and the timescales of lunar volcanism. If LRO is still active when any future mission reaches the surface, LRO's capability to rapidly place surface activities into broader geologic context will provide operational advantages. LRO remains a unique strategic asset that continues to address the needs of future missions

Building on the Cornerstone: Destinations for Nearside Sample Return

Discoveries from LRO (Lunar Reconnaissance Orbiter) have transformed our knowledge of the Moon, but LRO's instruments were originally designed to collect the measurements required to enable future lunar surface exploration. Compelling science questions and critical resources make the Moon a key destination for future human and robotic exploration. Lunar surface exploration, including rovers and other landed missions, must be part of a balanced planetary science and exploration portfolio. Among the highest planetary exploration priorities is the collection of new samples and their return to Earth for more comprehensive analysis than can be done in-situ. The Moon is the closest and most accessible location to address key science questions through targeted sample return. The Moon is the only other planet from which we have contextualized samples, yet critical issues need to be addressed: we lack important details of the Moon's early and recent geologic history, the full compositional and age ranges of its crust, and its bulk composition

Identifying and Characterizing Impact Melt Outcrops in the Nectaris Basin

The Nectaris Basin is an 820-km diameter, multi-ring impact basin located on the near side of the Moon. Nectaris is a defining stratigraphic horizon based on relationships between ejecta units, giving its name to the Nectarian epoch of lunar history. Lunar basin chronology based on higher resolution LRO imagery and topography, while assigning some important basins like Serenitatis to pre-Nectarian time, were generally consistent with those previously derived. Based on this stratigraphy, at least 11 large basins formed in the time between Nectaris and Imbrium. The absolute age of Nectaris, therefore, is a crucial marker in the lunar time-stratigraphic sequence for understanding the impact flux on the Moon, and by extension, the entire inner solar system. For several decades, workers have attempted to constrain the age of the Nectaris basin through radiometric dating of lunar samples. However, there is little agreement on which samples in our collection represent Nectaris, if any, and what the correct radiometric age of such samples is. The importance of the age of Nectaris goes far beyond assigning a stratigraphic marker to lunar chronology. Several dynamical models use Nectaris as their pin date, so that this date becomes crucial in understanding the time-correlated effects in the rest of the solar system. The importance of the Nectaris basin age, coupled with its nearside, mid-latitude location, make remnants of the impact-melt sheet an attractive target for a future mission, either for in-situ dating or for sample return. We have started exploring this possibility. We have begun a consortium data-analysis effort bringing multiple datasets and analysis methods to bear on these putative impact-melt deposits to characterize their extent, elemental composition and mineralogy, maturity and geologic setting, and to identify potential landing sites that meet both operational safety and science requirements

Profiling of dynamics in protein–lipid–water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths

Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes. Analysis of the time-resolved fluorescence spectroscopic data provides the characteristic time constant for the twisting motion of the BADAN label, which is sensitive to the local flexibility of the protein–lipid environment. In addition, we obtain information about the mobility of water molecules at the membrane–water interface. The results provide an unprecedented nanoscale profiling of the dynamics and distribution of water in membrane systems. This information gives clear evidence that the actual barrier of membranes for ions and aqueous solvents is located at the region of carbonyl groups of the acyl chains

Shape variability of the central sulcus in the developing brain: a longitudinal descriptive and predictive study in preterm infants

Despite growing evidence of links between sulcation and function in the adult brain, the folding dynamics, occurring mostly before normal-term-birth, is vastly unknown. Looking into the development of cortical sulci in infants can give us keys to address fundamental questions: what is the sulcal shape variability in the developing brain? When are the shape features encoded? How are these morphological parameters related to further functional development? In this study, we aimed to investigate the shape variability of the developing central sulcus, which is the frontier between the primary somatosensory and motor cortices. We studied a cohort of 71 extremely preterm infants scanned twice using MRI - once around 30 weeks post-menstrual age (w PMA) and once at term-equivalent age, around 40w PMA -, in order to quantify the sulcus's shape variability using manifold learning, regardless of age-group or hemisphere. We then used these shape descriptors to evaluate the sulcus's variability at both ages and to assess hemispheric and age-group specificities. This led us to propose a description of ten shape features capturing the variability in the central sulcus of preterm infants. Our results suggested that most of these features (8/10) are encoded as early as 30w PMA. We unprecedentedly observed hemispheric asymmetries at both ages, and the one captured at term-equivalent age seems to correspond with the asymmetry pattern previously reported in adults. We further trained classifiers in order to explore the predictive value of these shape features on manual performance at 5 years of age (handedness and fine motor outcome). The central sulcus's shape alone showed a limited but relevant predictive capacity in both cases. The study of sulcal shape features during early neurodevelopment may participate to a better comprehension of the complex links between morphological and functional organization of the developing brain