Better Understanding Of Structure-function Relationships Research Articles

Machine learning meets quantum mechanics in catalysis

Over the past decade many researchers have applied machine learning algorithms with computational chemistry and materials science tools to explore properties of catalysts. There is a rapid increase in publications demonstrating the use of machine learning for rational catalyst design. In our perspective, targeted tools for rational catalyst design will continue to make significant contributions. However, the community should focus on developing high-throughput simulation tools that utilize molecular dynamics capabilities for thorough exploration of the complex potential energy surfaces that exist, particularly in heterogeneous catalysis. Catalyst-specific databases should be developed to contain enough data to represent the complex multi-dimensional space that defines structure-function relationships. Machine learning tools will continue to impact rational catalyst design; however, we believe that more sophisticated pattern recognition algorithms would yield better understanding of structure-function relationships for heterogeneous catalysis.

The Mobility of the Cap Domain Is Essential for the Substrate Promiscuity of a Family IV Esterase from Sorghum Rhizosphere Microbiome

Metagenomics offers the possibility to screen for versatile biocatalysts. In this study, the microbial community of the Sorghum bicolor rhizosphere was spiked with technical cashew nut shell liquid, and after incubation, the environmental DNA (eDNA) was extracted and subsequently used to build a metagenomic library. We report the biochemical features and crystal structure of a novel esterase from the family IV, EH0, retrieved from an uncultured sphingomonad after a functional screen in tributyrin agar plates. EH0 (optimum temperature [Topt], 50°C; melting temperature [Tm], 55.7°C; optimum pH [pHopt], 9.5) was stable in the presence of 10 to 20% (vol/vol) organic solvents and exhibited hydrolytic activity against p-nitrophenyl esters from acetate to palmitate, preferably butyrate (496 U mg-1), and a large battery of 69 structurally different esters (up to 30.2 U mg-1), including bis(2-hydroxyethyl)-terephthalate (0.16 ± 0.06 U mg-1). This broad substrate specificity contrasts with the fact that EH0 showed a long and narrow catalytic tunnel, whose access appears to be hindered by a tight folding of its cap domain. We propose that this cap domain is a highly flexible structure whose opening is mediated by unique structural elements, one of which is the presence of two contiguous proline residues likely acting as possible hinges, which together allow for the entrance of the substrates. Therefore, this work provides a new role for the cap domain, which until now was thought to be an immobile element that contained hydrophobic patches involved in substrate prerecognition and in turn substrate specificity within family IV esterases. IMPORTANCE A better understanding of structure-function relationships of enzymes allows revelation of key structural motifs or elements. Here, we studied the structural basis of the substrate promiscuity of EH0, a family IV esterase, isolated from a sample of the Sorghum bicolor rhizosphere microbiome exposed to technical cashew nut shell liquid. The analysis of EH0 revealed the potential of the sorghum rhizosphere microbiome as a source of enzymes with interesting properties, such as pH and solvent tolerance and remarkably broad substrate promiscuity. Its structure resembled those of homologous proteins from mesophilic Parvibaculum and Erythrobacter spp. and hyperthermophilic Pyrobaculum and Sulfolobus spp. and had a very narrow, single-entry access tunnel to the active site, with access controlled by a capping domain that includes a number of nonconserved proline residues. These structural markers, distinct from those of other substrate-promiscuous esterases, can help in tuning substrate profiles beyond tunnel and active site engineering.

The vaccinologist\u2019s \u201cdirty little secret\u201d: a better understanding of structure-function relationships of viral immunogens might advance rational HIV vaccine design

I will offer a conceptual analysis of different notions of structure and function of viral immunogens and of different structure-function relationships. My focus will then be on the mechanisms by which the desired immune response is induced and why strategies based on three-dimensional molecular antigen structures and their rational design are limited in their ability to induce the desired immunogenicity. I will look at the mechanisms of action of adjuvants (thus the wordplay with Janeway’s “immunologist’s dirty little secret”). Strategies involving adjuvants and other (more successful) vaccination strategies rely on taking into account activities and functions (“what is going on”), and not just the structures involved (“who is there”), in binding in a “lock and key” fashion. Functional patterns as well as other organizational and temporal patterns, I will argue, are crucial for inducing the desired immune response and immunogenicity. The 3D structural approach by itself has its benefits – and its limits, which I want to highlight by this philosophical analysis, pointing out the importance of structure-function relationships. Different functional aspects such as antigenicity, immunogenicity, and immunity need to be kept separate and cannot be reduced to three-dimensional structures of vaccines. Taking into account different notions of structure and function and their relationships might thus advance our understanding of the immune system and rational HIV vaccine design, to which end philosophy can provide useful tools.

Characterization of permissive and non-permissive peptide insertion sites in chloramphenicol acetyltransferase

The growing prevalence of antibiotic resistance in numerous pathogenic bacteria is a major public health concern and urgently requires the development of new therapeutic approaches. Multidrug resistant species that remain sensitive to chloramphenicol (CAM) treatment have engendered renewed interest in using this drug as a modern day antimicrobial agent. High-level resistance to CAM commonly is mediated by chloramphenicol acetyltransferase (CAT) which catalyzes the acetylation of CAM and renders the drug inactive. Of the three main types (CATI, CATII and CATIII), CATI is of broad clinical significance. Despite this importance, understanding of the catalytic mechanism of CATI largely is extrapolated from studies of CATIII. Here, pentapeptide scanning mutagenesis was used to generate a library of random insertions in CATI to gain a better understanding of structure-function relationships in the enzyme. Pentapeptide insertions in secondary structure elements which contain residues that form part of the CATI active site abolished CAM resistance in Escherichia coli. Insertions in secondary structures that have key roles in protein folding and CAM binding led to a reduction in resistance. In contrast, insertions in loop regions between the major secondary structure features exerted modest, if any, effects on CAM resistance. The analysis pinpoints regions of CATI that may serve as targets for the design of novel inhibitors that prevent the spread of CAM-resistant pathogens thereby enabling the drug to be re-deployed as a broad range antimicrobial agent. Moreover, regions of CATI that are tolerant of insertions may be suitable for the construction of bifunctional enzymes in which peptides, mini-proteins or amino acid tags are introduced at the permissive sites.

RG-I galactan side-chains are involved in the regulation of the water-binding capacity of potato cell walls

Potato cell walls (PCW) are a low value by-product from the potato starch industry. Valorisation of PCW is hindered by its high water-binding capacity (WBC). The composition of polysaccharides and interactions between these entities, play important roles in regulating the WBC in the cell wall matrix. Here, we show that in vivo exo-truncation of RG-I β-(1→4)-D-galactan side-chains decreased the WBC by 6–9%. In contrast, exo-truncation of these side-chains increased the WBC by 13% in vitro. We propose that degradation of RG-I galactan side-chains altered the WBC of PCW, due to cell wall remodelling and loosening that affected the porosity. Our findings reinforce the view that RG-I galactan side-chains play a role in modulating WBC, presumably by affecting polysaccharide architecture (spacing) and interactions in the matrix. Better understanding of structure-function relationships of pectin macromolecules is needed before cell wall by-products may be tailored to render added-value in food and biobased products.

Antlers - Evolution, development, structure, composition, and biomechanics of an outstanding type of bone.

Antlers are bony appendages of deer that undergo periodic regeneration from the top of permanent outgrowths (the pedicles) of the frontal bones. Of the "less familiar" bone types whose study was advocated by John Currey to gain a better understanding of structure-function relationships of mineralized tissues and organs, antlers were of special interest to him. The present review summarizes our current knowledge about the evolution, development, structure, mineralization, and biomechanics of antlers and how their formation is affected by environmental factors like nutrition. Furthermore, the potential role of antlers as a model in bone biology and several fields of biomedicine as well as their use as a monitoring tool in environmental studies are discussed.

A novel thermophilic hemoprotein scaffold for rational design of biocatalysts.

Hemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze H2O2 decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by H2O2. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with H2O2, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design.

A Finite Element Bendo-Tensegrity Model of Eukaryotic Cell.

Mechanical interaction of cell with extracellular environment affects its function. The mechanisms by which mechanical stimuli are sensed and transduced into biochemical responses are still not well understood. Considering this, two finite element (FE) bendo-tensegrity models of a cell in different states are proposed with the aim to characterize cell deformation under different mechanical loading conditions: a suspended cell model elucidating the global response of cell in tensile test simulation and an adherent cell model explicating its local response in atomic force microscopy (AFM) indentation simulation. The force-elongation curve obtained from tensile test simulation lies within the range of experimentally obtained characteristics of smooth muscle cells (SMCs) and illustrates a nonlinear increase in reaction force with cell stretching. The force-indentation curves obtained from indentation simulations lie within the range of experimentally obtained curves of embryonic stem cells (ESCs) and exhibit the influence of indentation site on the overall reaction force of cell. Simulation results have demonstrated that actin filaments (AFs) and microtubules (MTs) play a crucial role in the cell stiffness during stretching, whereas actin cortex (AC) along with actin bundles (ABs) and MTs are essential for the cell rigidity during indentation. The proposed models quantify the mechanical contribution of individual cytoskeletal components to cell mechanics and the deformation of nucleus under different mechanical loading conditions. These results can aid in better understanding of structure-function relationships in living cells.

Gluten Polymer Networks-A Microstructural Classification in Complex Systems.

A classification of gluten polymer networks would support a better understanding of structure-function relationships of any gluten polymer material and thus, the control of processing properties. However, quantification and interpretation of the gluten network structures is challenging due to their complexity. Thus, the network formation was altered by specific gluten-modifying agents (glutathione, ascorbic acid, potassium bromate, glucose oxidase, transglutaminase, bromelain) in this study in order to clarify if structural alterations can be detected on a microstructural level and to specify different polymer arrangements in general. Microstructure analysis was performed by confocal laser scanning microscopy followed by quantification with protein network analysis. It was shown that alterations in gluten microstructure could be elucidated according to the kind of modification in cross-linking (disulphide, (iso) peptide, dityrosyl). Linear correlations of structural network attributes among each other were found, leading to an assertion in general: the higher the branching rate, the thinner the protein threads and the larger the interconnected protein aggregate. Considering the morphological attribute lacunarity, a quantitative classification of different gluten arrangements was established. These assertions were extended by using unspecific gluten-modifying agents in addition to the specific ones. Ultimately, five network types were proposed based on diverse polymer arrangements.

CRAC channel gating and its modulation by STIM1 and 2-aminoethoxydiphenyl borate.

Ca2+ release-activated Ca2+ (CRAC) channels play an essential role in the immune system. The pore-forming subunit, Orai1, is an important pharmacological target. Here, we summarize the recent discoveries on the structure-function relationship of Orai1, as well as its interaction with the native channel opener STIM1 and chemical modulator 2-aminoethoxydiphenyl borate (2-APB). We first introduce the critical structural elements of Orai1, which include a Ca2+ accumulating region, ion selectivity filter, hydrophobic centre, basic region, extended transmembrane Orai1 N-terminal (ETON) region, transmembrane (TM) regions 2 and 3, P245 bend, 263 SHK265 hinge linker and L273-L276 hydrophobic patch. We then hypothesize the possible mechanisms by which STIM1 triggers the conformational transitions of TM regions and exquisitely shapes the ion conduction pathway during generation of the CRAC current (Icrac ) with high Ca2+ selectivity. Finally, we propose mechanisms by which 2-APB modulates Icrac . On the STIM1-activated Orai1 channel, a low dose of 2-APB acts directly, dilating its extremely narrow pore diameter from 3.8 to 4.6Å, increasing its unitary channel conductance, and potentiating the Icrac . Further elucidation of the structure of the opened CRAC channel and a better understanding of structure-function relationship will benefit the future development of novel immune modulators.

Grey matter injury patterns in cerebral palsy: associations between structural involvement on MRI and clinical outcomes.

In a population cohort of children with grey matter injury (GMI) and cerebral palsy (CP), we aimed to describe and classify magnetic resonance imaging characteristics specific to GMI, and to identify key structure-function associations that serve as a basis for rating GMI in clinically relevant ways. Symmetry, extent of cerebral injury, and pathological pattern for 54 children (37 males, 17 females) with CP and a predominant GMI pattern on chronic-phase magnetic resonance imaging were related to gross motor function, motor type and topography, epilepsy, intellectual disability, blindness, and deafness. Relative to mild GMI where there was no pallidal abnormality, severe GMI, comprising pallidal abnormality alone or in conjunction with other deep nuclear and generalized cortical-subcortical involvement, was strongly associated with Gross Motor Function Classification System levels IV to V (OR 35.7 [95% CI 3.5, 368.8]). Involvement of the basal ganglia was associated with non-spastic/mixed motor types, but predominantly where cortical-subcortical grey and white matter involvement was not extensive. The prevalence of epilepsy was highest where there was diffuse cortical-subcortical involvement and white matter loss. Better understanding of structure-function relationships in CP and GMI, and how to rate the severity of GMI, will be helpful in the clinical context and also as a basis for investigation of causal pathways in CP.

Microarrays for identifying binding sites and probing structure of RNAs.

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.

The High-Resolution Structures of Two Piscidin Isoforms in Two Microbial Lipid Membrane Mimics Using Solid-State NMR

Piscidins 1 and 3 are twenty-two-residue-long amphipathic, cationic, antimicrobial peptides that have been isolated from hybrid striped bass. Since pathogens are becoming increasingly resistant to traditional antibiotics, antimicrobial peptides such as piscidin may prove to be useful in the future due to their broad-spectrum of activity against microbes and low induction of bacterial resistance. While membrane-active and fast-acting piscidins 1 and 3 have been shown to bind and disrupt bacterial lipid membrane mimics, their mode of action against microbes such as bacteria and yeasts is not known.In earlier studies, we used solid-state NMR to determine the three-dimensional atomic-resolution structures of piscidin 1 and piscidin 3 in aligned bilayers consisting of 3:1 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG). Here, the specific aim has been to solve the structures of these peptides in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) so that we could compare and contrast these structures with those in 3:1 DMPC/DMPG, a lipid system that has lower anionic content and does not exhibit the negative intrinsic membrane curvature of bacterial membranes. We have collected two-dimensional solid-state NMR spectra on 15N-backbone labeled piscidins in the presence of aligned 1:1 POPE/POPG bilayers. 15N chemical shifts and 15N-1H dipolar couplings have been combined to obtain the topological orientation of the peptides and calculate their atomic level backbone structures in the membrane-bound state. The data indicate that the carboxyl- and amino-ends of the peptides are affected differently by the changes in lipid composition. We will discuss the implications in terms of structural features and peptide-lipid interactions that may be important for piscidin's mode of action. Overall, these studies performed using native-like conditions provide us with a better understanding of structure-function relationships in piscidins and related antimicrobial peptides.

Special Issue: Retroviral Enzymes

The retroviral RNA genome encodes for three enzymes essential for virus replication: (i) the viral protease (PR), that converts the immature virion into a mature virus through the cleavage of precursor polypeptides; (ii) the reverse transcriptase (RT), responsible for the conversion of the single-stranded genomic RNA into double-stranded proviral DNA; and (iii) the integrase (IN) that inserts the proviral DNA into the host cell genome. All of them are important targets for therapeutic intervention. This Special Issue provides authoritative reviews on the most recent research towards a better understanding of structure-function relationships in retroviral enzymes. The Issue includes three reviews on retroviral PRs, seven on RT and reverse transcription, and four dedicated to viral integration. [...]

Molecular Aspects of Small Molecules-Poly(A) Interaction: An Approach to RNA Based Drug Design

The use of small molecules to specifically control important cellular functions is an area of major current interest at the interface of chemical biology and medicinal chemistry. Recognition of ribonucleic acids (RNA) has emerged more recently as a critical event in many biological pathways of eukaryotic cells and consequently the opportunity of drugs targeting to diverse structures of RNA is abundant. Such RNA targeting molecules must be able to specifically bind to unique structural organizations in RNA to regulate the gene expression. One particular example in this context is the modulation of the mRNA through its polyadenylic acid [poly(A)] tail. All mRNAs in eukaryotic cells have a poly(A) tail at the 3'-end This tail of about 200-250 or so adenine residues is an important determinant in maturation, stability of poly(A) and in initiation of translation process. Small molecules that could bind to this poly(A) tail could influence and possibly inhibit mRNA function and subsequent protein production in the cell leading to the development of new type of therapeutic agents. Recent discovery of the over expression of neo polyadenylic polymerase, the enzyme that catalyses polyadenylation, in human cancer cells compared to normal cells further signifies the importance of poly(A) in cellular events in cancer progression. The structural transition in poly(A) from single strand to double strand form induced by a narrow pH, salt and temperature variations also makes it a potential target for the better understanding of structure-function relationship in nucleic acids. Over the last forty years attempts have been made for the structural elucidation of this polyribonucleotide as well as the complex formed by the interactions with various small molecules like DNA intercalators, partial intercalators and groove binders using various physico-chemical and technique. These studies have led to progress in the understanding of specificity of binding, correlation between structural and thermodynamic aspects, description of drug-RNA binding modes and influence of substitutents on drug-RNA complexes and ultimately the discovery of new novel compounds that can be used as modulators of poly(A) structure. This review focuses on the structural and biological significance of poly(A), the use of small molecules to control the structure of this RNA and the futuristic development of new small molecules targeted to poly(A) structures.

Identification of a Single Tryptophan Residue as Critical for Binding Activity in a Humanized Monoclonal Antibody against Respiratory Syncytial Virus

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects.

Recent advances in egg protein functionality in the food system

Hen egg proteins are desirable ingredients of many foods. Today, eggs are widely distributed in international trade and the egg industry is an important segment of the world food industry. Hen egg proteins have unique functional properties such as gelling, foaming (egg white) and emulsifying (egg yolk) properties. This review describes the recent advances in molecular basis of the development of these functional properties as well as technical development of new approaches for improving the functional properties of egg proteins. A better understanding of structure-function relationship of egg protein functionality in food system will enable us for tailoring food protein for specific uses.

Exploiting expolysaccharides from lactic acid bacteria.

Microbial exopolysaccharides (EPSs) synthesized by lactic acid bacteria (LAB) play a major role in the manufacturing of fermented dairy products. EPS production is characterized by a large variety in terms of quantity, chemical composition, molecular size, charge, type of sidechains and rigidity of the molecules. Monosaccharide unit's composition, linkages, charge and size determine the EPS' intrinsic properties and their interactions with other milk constituents. EPSs contribute to texture, mouthfeel, taste perception and stability of the final product. Furthermore, it was reported that EPS from food grade organisms, particularly LAB, have potential as food additives and as functional food ingredients with both health and economic benefits. A better understanding of structure-function relationships of EPS in a dairy food matrix and of EPS biosynthesis remain two major challenges for further applications of EPS and the engineering of functional polysaccharides.

Structural requirements of tropomyosin for binding to filamentous actin.

Tropomyosin is an actin binding protein found in virtually all eucaryotic cells. Since its early discovery (Bailey, 1948), the actin binding properties and regulatory functions of tropomyosin have been extensively investigated. In addition, tropomyosin has served as a prototype for the structure of α-helical coiled-coil proteins (Cohen and Parry, 1990). The recent recognition of the diversity of tropomyosins in different cell types and discovery of the relationship between cell shape and isoform expression are indicative of a fundamental role for tropomyosin in the actin cytoskeleton. There is a need for a better understanding of structure-function relationships in this protein.

Insertion of transcriptional elements outside the replication region can interfere with replication, maintenance, and stability of Co1E1-derived plasmids.

The primary goal of our work is to contribute to a better understanding of structure-function relationships of nucleotide sequences defining promoters in theE. coli system. Since strong, unregulated promoters can be assumed to exhibit pertinent structural features most clearly, we investigated, in vitro, more than 50 promoters for their ability (a) to compete for the binding of RNA polymerase (an indication of their “signal strength”) and (b) to produce RNA (“overall efficiency” of a promoter) under conditions of limiting amounts of RNA polymerase. Promoters fulfilling these requirements best were found primarily in the genome of coliphage T5 (1). Some of these promoters show extremely high rates of complex formation with RNA polymerase (1,2) and at the same time were identified as being very efficient initiators of RNA synthesis in vitro and in vivo (1,3).KeywordsChloramphenicol Acetyl TransferaseTranscriptional SignalEfficient InitiatorDhfr GeneReplication RegionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.