A systematic study of the vibrational free energies of polypeptides in folded and random states

Molecular vibrations, especially low frequency motions, may be used as an indication of the rigidity or the flatness of the protein folding energy landscape. We have studied the vibrational properties of native folded as well as random coil structures of more than 60 polypeptides. The picture we obtain allows us to perceive how and why the energy landscape progressively rigidifies while still allowing potential flexibility. Compared with random coil structures, both alpha-helices and beta-hairpins are vibrationally more flexible. The vibrational properties of loop structures are similar to those of the corresponding random coil structures. Inclusion of an alpha-helix tends to rigidify peptides and so-called building blocks of the structure, whereas the addition of a beta-structure has less effect. When small building blocks coalesce to form larger domains, the protein rigidifies. However, some folded native conformations are still found to be vibrationally more flexible than random coil structures, for example, beta(2)-microglobulin and the SH3 domain. Vibrational free energy contributes significantly to the thermodynamics of protein folding and affects the distribution of the conformational substates. We found a weak correlation between the vibrational folding energy and the protein size, consistent with both previous experimental estimates and theoretical partition of the heat capacity change in protein folding.     

Allostery and conformational free energy changes in human tryptophanyl‐tRNA synthetase from essential dynamics and structure networks

The interdependence of the concept of allostery and enzymatic catalysis, and they being guided by conformational mobility is gaining increased prominence. However, to gain a molecular level understanding of allostery and hence of enzymatic catalysis, it is of utter importance that the networks of amino acids participating in allostery be deciphered. Our lab has been exploring the methods of network analysis combined with molecular dynamics simulations to understand allostery at molecular level. Earlier we had outlined methods to obtain communication paths and then to map the rigid/flexible regions of proteins through network parameters like the shortest correlated paths, cliques, and communities. In this article, we advance the methodology to estimate the conformational populations in terms of cliques/communities formed by interactions including the side‐chains and then to compute the ligand‐induced population shift. Finally, we obtain the free‐energy landscape of the protein in equilibrium, characterizing the free‐energy minima accessed by the protein complexes. We have chosen human tryptophanyl‐tRNA synthetase (hTrpRS), a protein responsible for charging tryptophan to its cognate tRNA during protein biosynthesis for this investigation. This is a multidomain protein exhibiting excellent allosteric communication. Our approach has provided valuable structural as well as functional insights into the protein. The methodology adopted here is highly generalized to illuminate the linkage between protein structure networks and conformational mobility involved in the allosteric mechanism in any protein with known structure. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Is landscape fragmentation always detrimental for species conservation? The case of the Iberian lynx in central Spain

The patch-corridor-matrix is the most commonly used model when dealing with landscape characterization studies, but it shows relevant limitations to detect landscape heterogeneity. Other authors have used a functional approach, since it is well known that nutrient, mineral and energy flows exist among ecosystems. These flows can be perceived in boundaries between different landcovers, making possible the identification of spatial units sharing a common pattern of ecological interactions known as mosaics. While the influence of each mosaic over a certain species has been previously addressed, no attention has been given to the intra-mosaic variation. The aim of this research is to assess the influence of functional diversity and connectivity, on the habitat suitability of the Iberian lynx (Lynx pardinus). For this, we built two GLMs to test if these features show a differential effect on lynxes’ habitat suitability depending on the mosaic. Both GLMs built show that the influence of these landscape features on lynxes’ habitat suitability depends on the landscape spatial organization and landcover composition, suggesting that there is no unique response of a species to changes in landscape diversity and/or connectivity. Thus, we conclude that considering both landscape and species features would allow to a better integration of land management strategies and conservation actions, which could favor species adaptation to highly human-modified landscapes.     

Examining the dynamic energy landscape of an antiporter upon inhibitor binding

Previously, we applied single-molecule force spectroscopy to detect and locate interactions within the functional Na⁺/H⁺ antiporter NhaA from Escherichia coli. It was observed that the binding of the inhibitor 2-aminoperimidine established interactions different from those introduced by the binding of the native ligand. To understand the inhibitory mechanism of the inhibitor, we applied single-molecule dynamic force spectroscopy to reconstruct the energy landscape of NhaA. Dynamic force spectroscopy revealed that the energy landscape of the antiporter remained mainly unchanged except for the energy barrier of the functionally important transmembrane α-helix IX. Inhibitor binding set this domain into a newly formed deep and narrow energy minimum that kinetically stabilized α-helix IX and reduced its conformational entropy. The entropy reduction of α-helix IX is thought to inhibit its functionally important structural flexibility, while the deeper energy barrier shifted the population of active antiporters towards inhibited antiporters.     

A Frustrated Binding Interface for Intrinsically Disordered Proteins

Intrinsically disordered proteins are very common in the eukaryotic proteome, and many of them are associated with diseases. Disordered proteins usually undergo a coupled binding and folding reaction and often interact with many different binding partners. Using double mutant cycles, we mapped the energy landscape of the binding interface for two interacting disordered domains and found it to be largely suboptimal in terms of interaction free energies, despite relatively high affinity. These data depict a frustrated energy landscape for interactions involving intrinsically disordered proteins, which is likely a result of their functional promiscuity.     

Role of extracellular glutamic acids in the stability and energy landscape of bacteriorhodopsin

Bacteriorhodopsin (BR), a specialized nanomachine, converts light energy into a proton gradient to power Halobacterium salinarum. In this work, we analyze the mechanical stability of a BR triple mutant in which three key extracellular residues, Glu⁹, Glu¹⁹⁴, and Glu²⁰⁴, were mutated simultaneously to Gln. These three Glu residues are involved in a network of hydrogen bonds, in cation binding, and form part of the proton release pathway of BR. Changes in these features and the robust photocycle dynamics of wild-type (WT) BR are apparent when the three extracellular Glu residues are mutated to Gln. It is speculated that such functional changes of proteins go hand in hand with changes in their mechanical properties. Here, we apply single-molecule dynamic force spectroscopy to investigate how the Glu to Gln mutations change interactions, reaction pathways, and the energy barriers of the structural regions of WT BR. The altered heights and positions of individual energy barriers unravel the changes in the mechanical and the unfolding kinetic properties of the secondary structures of WT BR. These changes in the mechanical unfolding energy landscape cause the proton pump to choose unfolding pathways differently. We suggest that, in a similar manner, the changed mechanical properties of mutated BR alter the functional energy landscape favoring different reaction pathways in the light-induced proton pumping mechanism.     

Computational delineation of the catalytic step of a high‐fidelity DNA polymerase

The Bacillus fragment, belonging to a class of high‐fidelity polymerases, demonstrates high processivity (adding ～115 bases per DNA binding event) and exceptional accuracy (1 error in 10⁶ nucleotide incorporations) during DNA replication. We present analysis of structural rearrangements and energetics just before and during the chemical step (phosphodiester bond formation) using a combination of classical molecular dynamics, mixed quantum mechanics molecular mechanics simulations, and free energy computations. We find that the reaction is associative, proceeding via the two‐metal‐ion mechanism, and requiring the proton on the terminal primer O3′ to transfer to the pyrophosphate tail of the incoming nucleotide before the formation of the pentacovalent transition state. Different protonation states for key active site residues direct the system to alternative pathways of catalysis and we estimate a free energy barrier of ～12 kcal/mol for the chemical step. We propose that the protonation of a highly conserved catalytic aspartic acid residue is essential for the high processivity demonstrated by the enzyme and suggest that global motions could be part of the reaction free energy landscape.     

Dynamical implications of Viral Tiling Theory

The Caspar-Klug classification of viruses whose protein shell, called viral capsid, exhibits icosahedral symmetry, has recently been extended to incorporate viruses whose capsid proteins are exclusively organised in pentamers. The approach, named ‘Viral Tiling Theory’, is inspired by the theory of quasicrystals, where aperiodic Penrose tilings enjoy 5-fold and 10-fold local symmetries. This paper analyses the extent to which this classification approach informs dynamical properties of the viral capsids, in particular the pattern of Raman active modes of vibrations, which can be observed experimentally.     

Energy–Landscape Integrated Analysis: A proposal for measuring complexity in internal agroecosystem processes (Barcelona Metropolitan Region, 1860–2000)

Farm systems are facing a global challenge amidst a socio-metabolic transition that places them in a dilemma between increasing land-use intensity to meet the growing demand of food, feed, fibres and fuels, while avoiding a biodiversity loss at the same time. To solve this dilemma a deeper research on how species richness is kept in different land-use patterns is required, according to the quantity and quality of the ecological disturbance that farmers carry out across the landscape. We propose an Energy–Landscape Integrated Analysis model that assesses both the complexity of internal energy loops, and the information held in the whole network of socio-metabolic energy fluxes, so as to correlate this energy-information interplay with the functional landscape structure. The results show that the landscape heterogeneity of Mediterranean land-use mosaics, created by traditional organic mixed-farming, have tended to vanish as a result of a simultaneous reduction in the complexity of the interlinking pattern of energy flows and the quantity of information carried by them. The model could help us to reveal how and why different agroecosystem managements lead to key turning points in the relationship of the energy profile with landscape ecological functioning. No doubt, these results will be very useful for designing more sustainable farm systems worldwide in the future.     

Effect of forest landscapes composition and configuration on bird community and its functional traits in a hotspot of biodiversity of Chile

Understanding the effect of landscape configuration on the bird species richness and their functional traits (dietary preferences) is important to link the conservation and restoration plans to the production of the crops. Our aims were: 1) to study the influence of forest types (native, mixed and plantations) on the bird species richness in two agroforestry landscapes (heterogeneous/homogeneous); 2) to assess the effect of size/density of forest patches in the birds’ functional traits; 3) to evaluate the effect of isolated trees on them, and 4) to discuss conservation and restoration measures for the birds’ functional traits in agroforestry landscapes. We used hierarchical occupancy models to evaluate the effect of different landscape metrics and detectability measures on bird communities. We recorded a total of 64 bird species. The estimated species richness was considerable higher in homogeneous landscape (31.7 ± 2.7) than heterogeneous (27.3 ± 2.5). Our results showed the bird assemblage had a positive trend with native forests, negative with mixed forests and neutral trend for plantations. The granivores and insectivore’s species showed significant preferences for homogeneous landscape, while omnivores had significant preferences for heterogeneous landscape. Carnivores/Piscivores and herbivores/frugivores species did not show preferences by any landscape type. The response of functional traits depended on different forests attributes. The isolated trees had a significant effect on the birds’ functional traits. In conclusion, it is necessary a deep knowledge about the relationship between the landscape configuration and the bird species richness/functional traits. These findings could help in the future with the conservation, restoration, and rewilding policies in this important hotspot of biodiversity, avoiding alterations in the ecosystem services.     

Functional biotic homogenization of bird communities in disturbed landscapes

Aim  Worldwide, functional homogenization is now considered to be one of the most prominent forms of biotic impoverishment induced by current global changes. Yet this process has hardly been quantified on a large scale through simple indices, and the connection between landscape disturbance and functional homogenization has hardly been established. Here we test whether changes in land use and landscape fragmentation are associated with functional homogenization of bird communities at a national scale.
Location  France.
Methods  We estimated functional homogenization of a community as the average specialization of the species present in that community. We studied the spatial variation of this community specialization index (CSI) using 1028 replicates from the French Breeding Bird Survey along spatial gradients of landscape fragmentation and recent landscape disturbance, measured independently, and accounting for spatial autocorrelation.
Results  The CSI was very sensitive to both measures of environmental degradation: on average, 23% of the difference in the CSI values between two sample sites was attributed to the difference in fragmentation and the disturbance between sites. This negative correlation between CSI and sources of landscape degradation was consistent over various habitats and biogeographical zones.
Main conclusions  We demonstrate that the functional homogenization of bird communities is strongly positively correlated to landscape disturbance and fragmentation. We suggest that the CSI is particularly effective for measuring functional homogenization on both local and global scales for any sort of organism and with abundance or presence–absence data.     

Dual energy landscape: the functional state of the β-barrel outer membrane protein G molds its unfolding energy landscape

We applied dynamic single-molecule force spectroscopy to quantify the parameters (free energy of activation and distance of the transition state from the folded state) characterizing the energy barriers in the unfolding energy landscape of the outer membrane protein G (OmpG) from Escherichia coli. The pH-dependent functional switching of OmpG directs the protein along different regions on the unfolding energy landscape. The two functional states of OmpG take the same unfolding pathway during the sequential unfolding of β-hairpins I-IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a rigidified extracellular gating loop L6. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.     

闽三角林地景观结构与功能连接度的空间耦合关系

以闽三角地区为例,为探讨景观连接度不同评价方法的科学性,提出一种基于最小耗费距离模型的成本距离指数表征景观功能连接度,并与传统的基于景观格局指数的景观结构连接度度量指标进行比较,揭示两者的空间耦合关系,为景观连接度的有效评价提供支持。结果表明：(1)2000—2020年,林地与其他土地利用类型之间发生不同程度的转化,林地转为建设用地370.72km²,占总转出面积的77.28%;草地和耕地转为林地80.45km²,占总转入面积的90.53%。(2)20年间,林地景观结构与功能连接度总体呈减小趋势,其中,湖里区林地景观结构与功能连接度降幅均最大,斑块密度、最大斑块指数、聚合度指数和斑块结合度指数分别下降了76.47%、51.33%、68.13%和68.40%,成本距离指数增加了178.56%。(3)成本距离指数与斑块密度、最大斑块指数、聚合度指数、斑块结合度指数和景观结构连接度综合指数均为负相关。(4)在研究期间,双变量局部空间自相关聚集类型由低-高转化为高-高,结构连接度无变化时,景观功能连接度降低;由高-高转化为低-高,结构连接度无变化时,...     

PolyUbiquitin Chain Linkage Topology Selects the Functions from the Underlying Binding Landscape

Ubiquitin (Ub) can generate versatile molecular signals and lead to different celluar fates. The functional poly-valence of Ub is believed to be resulted from its ability to form distinct polymerized chains with eight linkage types. To provide a full picture of ubiquitin code, we explore the binding landscape of two free Ub monomers and also the functional landscapes of of all eight linkage types by theoretical modeling. Remarkably, we found that most of the compact structures of covalently connected dimeric Ub chains (diUbs) pre-exist on the binding landscape. These compact functional states were subsequently validated by corresponding linkage models. This leads to the proposal that the folding architecture of Ub monomer has encoded all functional states into its binding landscape, which is further selected by different topologies of polymeric Ub chains. Moreover, our results revealed that covalent linkage leads to symmetry breaking of interfacial interactions. We further propose that topological constraint not only limits the conformational space for effective switching between functional states, but also selects the local interactions for realizing the corresponding biological function. Therefore, the topological constraint provides a way for breaking the binding symmetry and reaching the functional specificity. The simulation results also provide several predictions that qualitatively and quantitatively consistent with experiments. Importantly, the K48 linkage model successfully predicted intermediate states. The resulting multi-state energy landscape was further employed to reconcile the seemingly contradictory experimental data on the conformational equilibrium of K48-diUb. Our results further suggest that hydrophobic interactions are dominant in the functional landscapes of K6-, K11-, K33- and K48 diUbs, while electrostatic interactions play a more important role in the functional landscapes of K27, K29, K63 and linear linkages.     

Vibrations-determined properties of green fluorescent protein

The physicochemical characteristics of the green fluorescent protein (GFP), including the thermodynamic properties (entropy, enthalpy, Gibbs' free energy, heat capacity), normal mode vibrations, and atomic fluctuations, were investigated. The Gaussian 03 computational chemistry program was employed for normal mode analysis using the AMBER force field. The thermodynamic parameters and atomic fluctuations were then calculated from the vibrational eigenvalues (frequencies) and eigenvectors. The regions of highest rigidity were shown to be the beta-sheet barrel with the central alpha-helix, which bears the chromophore. The most flexible parts of the GFP molecule were the outlying loops that cover the top and bottom of the beta-barrel. This way, the balance between rigidity and flexibility is maintained, which is the optimal relationship for protein stability in terms of Gibbs' free energy. This dual-schemed structure satisfies the requirements for GFP function. In this sense, the structure of GFP resembles a nanoscale drum: a stiff cylinder with flexible vibrating end(s).     

A rugged free energy landscape separates multiple functional RNA folds throughout denaturation

The dynamic mechanisms by which RNAs acquire biologically functional structures are of increasing importance to the rapidly expanding fields of RNA therapeutics and biotechnology. Large energy barriers separating misfolded and functional states arising from alternate base pairing are a well-appreciated characteristic of RNA. In contrast, it is typically assumed that functionally folded RNA occupies a single native basin of attraction that is free of deeply dividing energy barriers (ergodic hypothesis). This assumption is widely used as an implicit basis to interpret experimental ensemble-averaged data. Here, we develop an experimental approach to isolate persistent sub-populations of a small RNA enzyme and show by single molecule fluorescence resonance energy transfer (smFRET), biochemical probing and high-resolution mass spectrometry that commitment to one of several catalytically active folds occurs unexpectedly high on the RNA folding energy landscape, resulting in partially irreversible folding. Our experiments reveal the retention of molecular heterogeneity following the complete loss of all native secondary and tertiary structure. Our results demonstrate a surprising longevity of molecular heterogeneity and advance our current understanding beyond that of non-functional misfolds of RNA kinetically trapped on a rugged folding-free energy landscape.     

Functional diversity of phyllostomid bats in an urban–rural landscape: A scale-dependent analysis

Urbanization is one of the most pervasive processes of landscape transformation, responsible for novel selection agents promoting functional community homogenization. Bats may persist in those environments, but the mechanisms responsible for their adaptability and the spatial scales in which the landscape imposes environmental filtering remain poorly studied in the Neotropics. We tested the hypothesis that landscape composition interacts with the spatial scale to affect the functional diversity of phyllostomids in an urban–rural gradient. Based on functional traits, we calculated indices of functional richness, divergence, evenness, and community-weighted means of morphological traits, and classified species into functional groups. We evaluated the changes in those variables in response to forest, grassland, and urbanized areas at 0.5, 1.25, and 2km scales. The number of functional groups, functional richness, and functional evenness tended to be higher in areas far from cities and with higher forest cover, whereas functional divergence increased in more urbanized areas. Our results show that the mean value of wing loading in the assemblage was negatively associated with landscape transformation at several spatial scales. However, environmental filtering driven by grass cover was particularly robust at the 0.5km scale, affecting big-sized species with long-pointed wings. Retaining natural forest in cattle ranging systems at ~12 km² appears to favor the functional evenness and number of functional groups of phyllostomids. Recognizing the scale of the effect on phyllostomid functional responses appears to be a fundamental issue for elucidating the spatial extent to which phyllostomid conservation planning in urban–rural landscapes should be addressed.