USE OF THE PHOTOGRAMMETRIC METHOD IN EARTH SCIENCES: EXAMPLE OF VARDZIA MUSEUM RESERVE

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Estimation of interdomain flexibility of N-terminus of factor H using residual dipolar couplings

Characterization of segmental flexibility is needed to understand the biological mechanisms of the very large category of functionally diverse proteins, exemplified by the regulators of complement activation, that consist of numerous compact modules or domains linked by short, potentially flexible, sequences of amino acid residues. The use of NMR-derived residual dipolar couplings (RDCs), in magnetically aligned media, to evaluate interdomain motion is established but only for two-domain proteins. We focused on the three N-terminal domains (called CCPs or SCRs) of the important complement regulator, human factor H (i.e. FH1-3). These domains cooperate to facilitate cleavage of the key complement activation-specific protein fragment, C3b, forming iC3b that no longer participates in the complement cascade. We refined a three-dimensional solution structure of recombinant FH1-3 based on nuclear Overhauser effects and RDCs. We then employed a rudimentary series of RDC datasets, collected in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids) under three different conditions, to estimate interdomain motions. This circumvents a requirement of previous approaches for technically difficult collection of five independent RDC datasets. More than 80% of conformers of this predominantly extended three-domain molecule exhibit flexions of < 40 °. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3), could facilitate recognition of C3b via initial anchoring and eventual reorganization of modules to the conformation captured in the previously solved crystal structure of a C3b:FH1-4 complex

Role of electrostatic interactions in amyloid beta-protein (Abeta) oligomer formation: A discrete molecular dynamics study

Pathological folding and oligomer formation of the amyloid beta-protein (Abeta) are widely perceived as central to Alzheimer's disease (AD). Experimental approaches to study Abeta self-assembly are problematic, because most relevant aggregates are quasi-stable and inhomogeneous. We apply a discrete molecular dynamics (DMD) approach combined with a four-bead protein model to study oligomer formation of the amyloid beta-protein (Abeta). We address the differences between the two most common Abeta alloforms, Abeta40 and Abeta42, which oligomerize differently in vitro. We study how the presence of electrostatic interactions (EIs) between pairs of charged amino acids affects Abeta40 and Abeta42 oligomer formation. Our results indicate that EIs promote formation of larger oligomers in both Abeta40 and Abeta42. The Abeta40 size distribution remains unimodal, whereas the Abeta42 distribution is trimodal, as observed experimentally. Abeta42 folded structure is characterized by a turn in the C-terminus that is not present in Abeta40. We show that the same C-terminal region is also responsible for the strongest intermolecular contacts in Abeta42 pentamers and larger oligomers. Our results suggest that this C-terminal region plays a key role in the formation of Abeta42 oligomers and the relative importance of this region increases in the presence of EIs. These results suggest that inhibitors targeting the C-terminal region of Abeta42 oligomers may be able to prevent oligomer formation or structurally modify the assemblies to reduce their toxicity.Comment: Accepted for publication at Biophysical Journa

New insights into complex social organization in the southern Caucasus – Late Bronze Age–Early Iron Age settlement patterns in the Shiraki Plain (southeast Georgia)

Due to its location between Mesopotamia and the Eurasian steppes, the southern Caucasus occupies a distinctive place in Old World archeology. While several local areas in the South Caucasus have shown a complex social organization with fortified structures during the Late Bronze Age and Early Iron Age (LBA–EIA), the Shiraki Plain (southeast Georgia), despite its immense potential, has yet to be examined in a regional context. The presented research aims to conduct an initial multi-stage landscape archeological survey over the Shiraki Plain in order to reveal the LBA–EIA fortified settlements of previously identified Didnauri and Nazarlebi types. The authors use remote sensing, photogrammetry, and terrestrial survey methods to investigate selected areas. The results demonstrate the density and complexity of the massive, fortified structures spread over both the plain and neighboring ridges. Their spatial distribution suggests the idea of a well-organized defensive system adapted to the landscape, which in turn is discussed in a broader regional context.</p

Human-environmental interactions and seismic activity in a Late Bronze to Early Iron Age settlement center in the southeastern Caucasus

Long-term human-environmental interactions in naturally fragile drylands are a focus of geomorphological and geoarchaeological research. Furthermore, many dryland societies were also affected by seismic activity. The semi-arid Shiraki Plain in the tectonically active southeastern Caucasus is currently covered by steppe and largely devoid of settlements. However, numerous Late Bronze to Early Iron Age city-type settlements suggest early state formation between ca. 3.2-2.5 ka that abruptly ended after that time. A paleolake was postulated for the lowest plain, and nearby pollen records suggest forest clearcutting of the upper altitudes under a more humid climate during the Late Bronze/Early Iron Ages. Furthermore, also an impact of earthquakes on regional Early Iron Age settlements was suggested. However, regional paleoenvironmental changes and paleoseismicity were not systematically studied so far. We combined geomorphological, sedimentological, chronological and paleoecological data with hydrological modelling to reconstruct regional Holocene paleoenvironmental changes, to identify natural and human causes and to study possible seismic events during the Late Bronze/Early Iron Ages. Our results show a balanced to negative Early to Mid-Holocene water balance probably caused by forested upper slopes. Hence, no lake but a pellic Vertisol developed in the lowest plain. Following, Late Bronze/Early Iron Age forest clear-cutting caused lake formation and the deposition of lacustrine sediments derived from soil erosion. Subsequently, regional aridification caused slow lake desiccation. Remains of freshwater fishes indicate that the lake potentially offered valuable ecosystem services for regional prehistoric societies even during the desiccation period. Finally, colluvial coverage of the lake sediments during the last centuries could have been linked with hydrological extremes during the Little Ice Age. Our study demonstrates that the Holocene hydrological balance of the Shiraki Plain was and is situated near a major hydrological threshold, making the landscape very sensitive to small-scale human or natural influences with severe consequences for local societies. Furthermore, seismites in the studied sediments do not indicate an influence of earthquakes on the main and late phases of Late Bronze/Early Iron Age settlement. Altogether, our study underlines the high value of multi-disciplinary approaches to investigate human-environmental interactions and paleoseismicity in drylands on millennial to centennial time scales

The Bronze Age occupation of the Black Sea coast of Georgia—New insights from settlement mounds of the Colchian plain

Along the lower course of the Rioni and several minor rivers, more than 70 settlement mounds (local name: Dikhagudzuba) have been identified by field surveys and remote sensing techniques. They give evidence of a formerly densely populated landscape in the coastal lowlands on the Colchian plain (western Georgia) and have been dated to the Bronze Age. As yet, limited information is available on their internal architecture, the chronology of the different layers and their palaeoenvironmental context. Based on archaeological sources, remote sensing measurements of three mounds and sediment cores from one mound and its closer surroundings, our study presents a review of the relevant literature and reveals the internal structure, distribution and spatial extent of the mounds. Geochemical and sedimentological analyses of element contents (X‐ray fluorescence) and granulometry helped to identify different stratigraphical layers and differentiate between natural facies and anthropogenic deposits; using the Structure‐from‐Motion technique the mounds' dimensions were calculated. The studied settlement mounds had relatively small dimension (varying from 30 to 100 m in diameter) and were similar in their stratigraphy. Measurement of elements that can identify types of human activity, notably metals and phosphorus, suggest changing intensities of human occupation, pastoral agriculture and metalworking through the occupation sequence. According to the 14C chronology, the formation of the settlements occurred during the first half of the second millennium B.C., which confirms the archaeological interpretation of their Bronze Age origin. The narrow age difference between the lowermost and uppermost anthropogenic layers indicates an intentional construction of the mounds, rather than a successive accumulation of construction debris due to the disintegration of loam bricks by weathering. Therefore, they are indeed mounds and not tells. It is most likely that the characteristic circular moats that surround them were the source of their construction material. Fluvial and alluvial processes in a warm and humid climate dominated the environment of the mounds

Use of time-resolved FRET to validate crystal structure of complement regulatory complex between C3b and factor H (N terminus)

Structural knowledge of interactions amongst the ∼ 40 proteins of the human complement system, which is central to immune surveillance and homeostasis, is expanding due primarily to X-ray diffraction of co-crystallized proteins. Orthogonal evidence, in solution, for the physiological relevance of such co-crystal structures is valuable since intermolecular affinities are generally weak-to-medium and inter-domain mobility may be important. In this current work, Förster resonance energy transfer (FRET) was used to investigate the 10 μM K(D) (210 kD) complex between the N-terminal region of the soluble complement regulator, factor H (FH1-4), and the key activation-specific complement fragment, C3b. Using site-directed mutagenesis, seven cysteines were introduced individually at potentially informative positions within the four CCP modules comprising FH1-4, then used for fluorophore attachment. C3b possesses a thioester domain featuring an internal cycloglutamyl cysteine thioester; upon hydrolysis this yields a free thiol (Cys988) that was also fluorescently tagged. Labeled proteins were functionally active as cofactors for cleavage of C3b to iC3b except for FH1-4(Q40C) where conjugation with the fluorophore likely abrogated interaction with the protease, factor I. Time-resolved FRET measurements were undertaken to explore interactions between FH1-4 and C3b in fluid phase and under near-physiological conditions. These experiments confirmed that, as in the cocrystal structure, FH1-4 binds to C3b with CCP module 1 furthest from, and CCP module 4 closest to, the thioester domain, placing subsequent modules of FH near to any surface to which C3b is attached. The data do not rule out flexibility of the thioester domain relative to the remainder of the complex

Soluble oligomers are sufficient for transmission of a yeast prion but do not confer phenotype

Amyloidogenic proteins aggregate through a self-templating mechanism that likely involves oligomeric or prefibrillar intermediates. For disease-associated amyloidogenic proteins, such intermediates have been suggested to be the primary cause of cellular toxicity. However, isolation and characterization of these oligomeric intermediates has proven difficult, sparking controversy over their biological relevance in disease pathology. Here, we describe an oligomeric species of a yeast prion protein in cells that is sufficient for prion transmission and infectivity. These oligomers differ from the classic prion aggregates in that they are soluble and less resistant to SDS. We found that large, SDS-resistant aggregates were required for the prion phenotype but that soluble, more SDS-sensitive oligomers contained all the information necessary to transmit the prion conformation. Thus, we identified distinct functional requirements of two types of prion species for this endogenous epigenetic element. Furthermore, the nontoxic, self-replicating amyloid conformers of yeast prion proteins have again provided valuable insight into the mechanisms of amyloid formation and propagation in cells

Molecular Structure of Amyloid Fibrils Controls the Relationship between Fibrillar Size and Toxicity

According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.For one amyloid state, fibril fragmentation was found to enhance its cytotoxic potential, whereas for another amyloid state formed within the same amino acid sequence, the fragmented fibrils were found to be substantially less toxic than the intact fibrils. Consistent with the previous studies, the toxic effects were more pronounced for cell cultures expressing normal isoform of the prion protein (PrP(C)) at high levels confirming that cytotoxicity was in part PrP(C)-dependent. Silencing of PrP(C) expression by small hairpin RNAs designed to silence expression of human PrP(C) (shRNA-PrP(C)) diminished the deleterious effects of the two amyloid states to a different extent, suggesting that the role of PrP(C)-mediated and PrP(C)-independent mechanisms depends on the structure of the aggregates.This work provides a direct illustration that the relationship between an amyloid's physical dimension and its toxic potential is not unidirectional but is controlled by the molecular structure of prion protein (PrP) molecules within aggregated states. Depending on the structure, a decrease in size of amyloid fibrils can either enhance or abolish their cytotoxic effect. Regardless of the molecular structure or size of PrP aggregates, silencing of PrP(C) expression can be exploited to reduce their deleterious effects

The chicken–egg scenario of protein folding revisited

AbstractWhat is the first step in protein folding – hydrophobic collapse (compaction) or secondary structure formation? It is still not clear if the major driving force in protein folding is hydrogen bonding or hydrophobic interactions or both. We analyzed data on the conformational characteristics of 41 globular proteins in native and partially folded conformational states. Our analysis shows that a good correlation exists between relative decrease in hydrodynamic volume and increase in secondary structure content. No compact equilibrium intermediates lacking secondary structure, or highly ordered non-compact species, were found. This correlation provides experimental support for the hypothesis that hydrophobic collapse occurs simultaneously with formation of secondary structure in the early stages of the protein folding