Secondary Structural Features Research Articles

Genotype-protein phenotype characterization of NOD2 and IL23R missense variants associated with inflammatory bowel disease: A paradigm from molecular modelling, dynamics, and docking simulations.

Inflammatory bowel disease (IBD) is a gastrointestinal disease with an underlying contribution of genetic, microbial, environment, immunity factors. The coding region risk markers identified by IBD genome wide association studies have not been well characterized at protein phenotype level. Therefore, this study is conducted to characterize the role of NOD2 (Arg675Trp and Gly908Arg) and IL23R (Gly149Arg and Arg381Gln) missense variants on the structural and functional features of corresponding proteins. Thus, we used different variant pathogenicity assays, molecular modelling, secondary structure, stability, molecular dynamics, and molecular docking analysis methods. Our findings suggest that SIFT, Polyphen, GREP++, PhyloP, SiPhy and REVEL methods are very sensitive in determining pathogenicity of NOD2 and IL23R missense variants. We have also noticed that all the tested missense variants could potentially alter secondary (α-helices, β-strands, and coils) and tertiary (residue level deviations) structural features. Moreover, our molecular dynamics (MD) simulation findings have simulated that NOD2 (Arg675Trp and Gly908Arg) and IL23R (Gly149Arg and Arg381Gln) variants creates rigid local structures comprising the protein flexibility and conformations. These predictions are corroborated by molecular docking results, where we noticed that NOD2 and IL23R missense variants induce molecular interaction deformities with RIPK2 and JAK2 ligand molecules, respectively. These functional alterations could potentially alter the signal transduction pathway cascade involved in inflammation and autoimmunity. Drug library searches and findings from docking studies have identified the inhibitory effects of Tacrolimus and Celecoxib drugs on NOD2 and IL23R variant forms, underlining their potential to contribute to personalized medicine for IBD. The present study supports the utilization of computational methods as primary filters (pre-in vitro and in vivo) in studying the disease potential mutations in the context of genptype-protein phenotype characteristics.

Exploiting natural riboswitches for aptamer engineering and validation.

Over the past three decades, researchers have found that some engineered aptamers can be made to work well in test tubes but that these same aptamers might fail to function in cells. To help address this problem, we developed the 'Graftamer' approach, an experimental platform that exploits the architecture of a natural riboswitch to enhance in vitro aptamer selection and accelerate in vivo testing. Starting with combinatorial RNA pools that contain structural features of a guanine riboswitch aptamer interspersed with regions of random sequence, we performed multiplexed in vitro selection with a collection of small molecules. This effort yielded aptamers for quinine, guanine, and caffeine that appear to maintain structural features of the natural guanine riboswitch aptamer. Quinine and caffeine aptamers were each grafted onto a natural guanine riboswitch expression platform and reporter gene expression was monitored to determine that these aptamers function in cells. Additionally, we determined the secondary structure features and survival mechanism of a class of RNA sequences that evade the intended selection strategy, providing insight into improving this approach for future efforts. These results demonstrate that the Graftamer strategy described herein represents a convenient and straightforward approach to develop aptamers and validate their in vivo function.

Influence of the Enzymatic Hydrolysis Using Flavourzyme Enzyme on Functional, Secondary Structure, and Antioxidant Characteristics of Protein Hydrolysates Produced from Bighead Carp (Hypophthalmichthys nobilis)

In the current study, bighead carp fish were used in conjunction with the flavourzyme enzyme to obtain (FPH) fish protein hydrolysates. The optimum conditions of the hydrolysis process included an enzyme/substrate ratio of 4% and a temperature of 50 °C and pH of 6.5. The hydrolysis time was studied and investigated at 1, 3, and 6 h, and the (DH) degree of hydrolysis was recorded at 16.56%, 22.23%, and 25.48%, respectively. The greatest yield value was 17.83% at DH 25.48%. By increasing the DH up to 25.48%, the crude protein and total amino acid composition of the hydrolysate were 88.19% and 86.03%, respectively. Moreover, more peptides with low molecular weight were formed during hydrolysis, which could enhance the functional properties of FPH, particularly the solubility property ranging from 85% to 97%. FTIR analysis revealed that enzymatic hydrolysis impacted the protein's secondary structure, as indicated by a remarkable wavelength of amide bands. Additionally, antioxidant activities were investigated and showed high activity of DDPH radical scavenging, and hydroxyl radical scavenging demonstrated remarkable activity. The current findings demonstrate that the functional, structural, and antioxidant characteristics of FPH might make it an excellent source of protein and suggest potential applications in the food industry.

Rtools: A Web Server for Various Secondary Structural Analyses on Single RNA Sequences.

Predicting the secondary structures of RNA molecules is an essential step to characterize their functions, but the thermodynamic probability of any prediction is generally small. On the other hand, there are a few tools for calculating and visualizing various secondary structural information from RNA sequences. We implemented a web server that calculates in parallel various features of secondary structures: different types of secondary structure predictions, the marginal probabilities for local structural contexts, accessibilities of the subsequences, the energy changes by arbitrary base mutations, and the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp , which integrates software tools, CentroidFold, CentroidHomfold, IPknot, CapR, Raccess, Rchange, RintD, and RintW.

DNA fragility at the KMT2A/MLL locus: insights from old and new technologies.

The Mixed-Lineage Leukaemia (MLL/KMT2A) gene is frequently rearranged in childhood and adult acute leukaemia (AL) and in secondary leukaemias occurring after therapy with DNA topoisomerase targeting anti-cancer agents such as etoposide (t-AL). MLL/KMT2A chromosome translocation break sites in AL patients fall within an 8 kb breakpoint cluster region (BCR). Furthermore, MLL/KMT2A break sites in t-AL frequently occur in a much smaller region, or hotspot, towards the 3' end of the BCR, close to the intron 11/exon 12 boundary. These findings have prompted considerable effort to uncover mechanisms behind the apparent fragility of the BCR and particularly the t-AL hotspot. Recent genome-wide analyses have demonstrated etoposide-induced DNA cleavage within the BCR, and it is tempting to conclude that this cleavage explains the distribution of translocation break sites in t-AL. However, the t-AL hotspot and the centre of the observed preferential DNA cleavage are offset by over 250 nucleotides, suggesting additional factors contribute to the distribution of t-AL break sites. We review these recent genomic datasets along with older experimental results, analysis of TOP2 DNA cleavage site preferences and DNA secondary structure features that may lead to break site selection in t-AL MLL/KMT2A translocations.

Direct imaging of intracellular RNA, DNA, and liquid–liquid phase separated membraneless organelles with Raman microspectroscopy

Methodologies for direct intracellular imaging of RNA and DNA are necessary for the advancement of bioimaging. Here we show direct label-free imaging of RNA and DNA in single cells by isolating their accurate Raman spectra. Raman images of DNA from interphase cells show intact nucleus, while those from mitotic cells reveal condensed chromosome. The condensed chromosome images are accurate enough to assign the stage of mitotic cell division (e.g., metaphase). Raman spectral features indicate B-DNA double helical conformational form in all the cell lines investigated here. The Raman images of RNAs, on the other hand, reveal liquid-liquid phase separated (LLPS) membraneless organelles in interphase cells, which disappears during mitosis. Further, the Raman spectrum of proteins from the intracellular LLPS organelles indicates slight enrichment of amyloid-like secondary structural features. Vibrational imaging of intracellular DNA and RNA simultaneously would open myriad of opportunities for examining functional biochemical aspects of cells and organelles.

Characterization of the complete mitogenome of Trachylophus sinensis (Coleoptera: Cerambycidae: Cerambycinae), the type species of Trachylophus and its phylogenetic implications

• The first complete mitogenome from the genus Trachylophus was determined using next-generation sequencing. • All available mitogenome of Cerambycinae were comparative and analysis to better understand of their mitogenomic evolution patterns. • The phylogenetic relationship of Cerambycinae was explored through all available mitogenome downloaded from GenBank. Complete mitochondrial genomes (mitogenomes) have long been proved as reliable markers for phylogenetic reconstruction among diverse animal groups, especially benefited from recent rapid development of sequencing techniques. However, the mitogenomes of many important clades remain poorly represented, which restricted the understanding of macroscale evolutionary history of these groups. Here, we sequenced and characterized the complete mitogenome of Trachylophus sinensis , a type species of the Trachylophus genus, which also represents the first sequenced mitogenome in this genus. The complete circular mitogenome was 15,746 bp in length, containing 37 typical genes and one noncoding AT-rich control region. The nucleotide composition of the mitogenome was highly A + T biased, accounting for 70.07% of the whole mitogenome with a slightly positive AT skewness (0.106). The 13 Protein coding genes (PCGs) used ATN as their start codons, except nad1 which used TTG. All tRNA genes were predicted with a characteristic cloverleaf secondary structure except trnS1 (AGN) , whose dihydrouridine (DHU) arm was replaced by a simple loop. Phylogenetic analyses recovered Cerambycinae as a monophyletic group with high node supports and the sister relationship between T. sinensis and Nadezhdiella cantori . However, we found that deeper nodes showed not strong support, which may be caused by limited taxa sampling in our study. More mitogenomes should be sequenced representing various taxonomic levels, especially closely related species, which will enhance our understanding of phylogenetic relationships among Cerambycinae.

Structural characterization and conformational dynamics of alpha-1 antitrypsin pathogenic variants causing alpha-1-antitrypsin deficiency.

Background: Alpha-1 antitrypsin deficiency (A1ATD) is a progressive lung disease caused by inherited pathogenic variants in the SERPINA1 gene. However, their actual role in maintenance of structural and functional characteristics of the corresponding α-1 anti-trypsin (A1AT) protein is not well characterized. Methods: The A1ATD causative SERPINA1 missense variants were initially collected from variant databases, and they were filtered based on their pathogenicity potential. Then, the tertiary protein models were constructed and the impact of individual variants on secondary structure, stability, protein-protein interactions, and molecular dynamic (MD) features of the A1AT protein was studied using diverse computational methods. Results: We identified that A1ATD linked SERPINA1 missense variants like F76S, S77F, L278P, E288V, G216C, and H358R are highly deleterious as per the consensual prediction scores of SIFT, PolyPhen, FATHMM, M-CAP and REVEL computational methods. All these variants were predicted to alter free energy dynamics and destabilize the A1AT protein. These variants were seen to cause minor structural drifts at residue level (RMSD = <2Å) of the protein. Interestingly, S77F and L278P variants subtly alter the size of secondary structural elements like beta pleated sheets and loops. The residue level fluctuations at 100ns simulation confirm the highly damaging structural consequences of all the six missense variants on the conformation dynamics of the A1AT protein. Moreover, these variants were also predicted to cause functional deformities by negatively impacting the binding energy of A1AT protein with NE ligand molecule. Conclusion: This study adds a new computational biology dimension to interpret the genotype-protein phenotype relationship between SERPINA1 pathogenic variants with its structural plasticity and functional behavior with NE ligand molecule contributing to the Alpha-1-antitrypsin deficiency. Our results support that A1ATD complications correlates with the conformational flexibility and its propensity of A1AT protein polymerization when misfolded.

A portable elliptical dichroism spectrometer targeting secondary structural features of tumorous protein for pancreatic cancer detection

Stereochemical analysis is essential for understanding the complex function of biomolecules. Various direct and indirect approaches can be used to explore the allosteric configuration. However, the size, cost, and delicate nature of these systems limit their biomedical usage. Here, we constructed elliptical dichroism (ED) spectrometer for biomedical applications, whose performance is validated by experiment and theoretical simulation (Jones/Mueller calculus and time-dependent density-functional theory). Instead of complicated control of circular polarization, ED spectrometer adopted the absorbance of left- and right-oriented elliptically polarized light. With a simplified design, we demonstrated the potential of ED spectrometry as an alternative for secondary structural analysis of biomolecules, their conformation and chirality. It not only provides a portable, low-cost alternative to the sophisticated instruments currently used for structural analysis of biomolecules but also provides superior translational features: low sample consumption(200 μl), easy operation, and multiple working modes, for noninvasive cancer detection.

Secondary flow structure characteristics of an automotive mixed flow turbocharger turbine volute at different aspect ratios

Secondary flow structure characteristics of an automotive mixed flow turbocharger turbine volute at different aspect ratios

A biophysical study of DNA condensation mediated by histones and protamines

The compaction of long DNA strands into confined spaces such as the nuclei of eukaryotic cells is an essential phenomenon towards the emergence of elaborated forms of life. Histones and protamines are the major nucleoproteins involved in this task participating in the formation of chromatin in somatic and germinative cells, respectively. In addition to a fundamental understanding of critical biological processes, DNA condensation also holds strong potential in biotechnology. Herein, we investigate the mesoscale structure of complexes formed between DNA and histones or protamines. A sophisticated set of biophysical methods encompassing steady-state fluorimetry, small-angle X-ray scattering and infrared nano spectroscopy was used to unveil both the self-assembly and molecular interactions of these complexes. We explored the fluorescence of a molecular rotor, thioflavin T, to investigate the accessibility of ligands in the inter-base environment of DNA strands. AFM-based infrared spectroscopy was used for the first time to probe the vibrational signature of individual DNA/nucleoprotein nano assemblies and disclose secondary-structure features. Our results show that protamines form highly compact structures in which DNA folding hinders access to the inter-base spacing. These assemblies exhibit diversified secondary-structure conformations, with the presence of β-sheets stabilizing the packing. In contrast, histone-based complexes are characterized by fibrillar nano assemblies exhibiting larger inter strands separations and access to guest molecules that intercalate between bases. The findings presented here may help the understanding of DNA condensation mediated by these two major nucleoproteins and may assist the optimization of gene vehicles based on these promising nano assemblies.

Microstructure and crystal structure dependence of microwave dielectric properties of non-stoichiometric (Sr0.7Ca0.3)z(Zr0.95Ti0.05)O3 perovskite ceramics

Solid-state reaction process has been used to fabricate (Sr0.7Ca0.3)z(Zr0.95Ti0.05)O3 (0.98 ≤ z ≤ 1.04) ceramics, the effects of non-stoichiometric ratio on sintering behavior, microstructure, structural properties and microwave dielectric properties were investigated. The ceramics contain the secondary phase ZrO2 with Fm-3m and P42 space structure as z ≤ 1.01. Electron back scatter diffraction analysis indicates that the secondary phase is concentrated at the grain boundary, which hinders the grain growth. Based on Rietveld refinement and Raman spectra, the secondary phase content and structural characteristics, including cell parameters, polarizability and lattice vibration anharmonicity, are obtained to establish the correlation between structure and microwave dielectric properties. The relative permittivity depends on the polarizability, and the variation of Q × f values is determined by the combined effects of grain size, secondary phase and lattice intrinsic loss, and is mainly driven by lattice vibration anharmonicity. In addition, the τf values are affected by lattice distortion. Particularly, (Sr0.7Ca0.3)1.02(Zr0.95Ti0.05)O3 (z = 1.02) ceramics have the optimized microwave dielectric properties: εr = 35.9, Q × f = 26,000 GHz, τf = −13 ppm/°C. This work provides a reference for optimizing microwave dielectric properties of materials.

Iodenium or Phosphonium: The Ambi-Valent Character of Iodophosphonium Complexes.

The ambi-valent character of the P-I bond in iodophosphonium complexes ensures that it can be electrophilic at either P or I. Herein, we use an ensemble of computational tools and methodologies to probe the nature of this ambi-valent bond. Geometric and atomic electron population analyses yielded strong trends between the electron donating ability of the phosphine and the strength and polarity of the P-I bond. Quasi-atomic orbital analysis demonstrated the near homo-polarity of the P-I bond, and energy decomposition analysis calculations demonstrated the ability to tune the polarization of the bond with only mild changes in secondary structural features. Finally, the ambi-valent nature of the P-I bond was demonstrated to follow hard-soft considerations in reactions with nucleophiles, with harder nucleophiles preferentially forming products of addition to P and softer nucleophiles to I.

Tuning Xanthan Viscosity by Directed Random Coil-to-Helix Transition.

Xanthan gum is a polysaccharide that is widely used as a thickening agent in numerous food, cosmetic, and technical applications. Therefore, the knowledge of the molecular interplay that builds up and stabilizes water-binding networks is crucial for the optimization of xanthan thickening performance. Using atomic force microscopy, rheometry, and inductively coupled plasma optical emission spectroscopy, we show a clear correlation between xanthan thickening properties and the ability to form characteristic secondary structures as well as the valence and amount of cations coordinated at the polysaccharide side chain. Based on these findings and the Debye-Hückel theory, we derive an ion-interaction model in which divalent cations mediate bridging of adjacent single polymer strands due to chelate-like coordination building stable secondary structures. We furthermore demonstrate in a cation exchange assay that xanthan secondary structures can be modified in a directed and reversible manner, which, in turn, alters its thickening properties.

Understanding the mechanism of amylin aggregation: From identifying crucial segments to tracing dominant sequential events to modeling potential aggregation suppressors

One of the most abundant, prevailing, and life-threatening human diseases that are currently baffling the scientific community is type 2 diabetes (T2D). The self-association of human amylin has been implicated in the pathogenesis of T2D, though with an inconclusive understanding of the mechanism. Hence, we focused on the characterization of the conformational ensembles of all the species that are believed to define the structural polymorphism of the aggregation process – the functional monomeric, the initially self-associated oligomeric, and the structured protofibril – by employing near-equilibrium, non-equilibrium, and equilibrium atomistic simulations on the sporadic, two familial variants (S20G and G33R), and their proline-substituted forms (S20P and G33P). The dynamic near-equilibrium assays hint toward – the abundance of helical conformation in the monomeric state, the retainment of the helicity in the initial self-associated oligomeric phase pointing toward the existence of the helix-helix association mechanism, the difference in preference of specific segments to have definite secondary structural features, the phase-dependent variability in the dominance of specific segments and mutation sites, and the simultaneous presence of generic and unique features among various sequences. Furthermore, the non-equilibrium pulling assays exemplify a generic sequential unzipping mechanism of the protofibrils, however, the sequence-dependent uniqueness comes from the difference in location and magnitude of the control of a specific terminus. Importantly, the equilibrium thermodynamic assays efficiently rank order the potential of aggregability among sequences and consequently suggests the probability of designing effective aggregation suppressors against sporadic and familial amylin variants incorporating proline as the mutation.

Identification of the genes at S and Z reveals the molecular basis and evolution of grass self-incompatibility.

Self-incompatibility (SI) is a feature of many flowering plants, whereby self-pollen is recognized and rejected by the stigma. In grasses (Poaceae), the genes controlling this phenomenon have not been fully elucidated. Grasses have a unique two-locus system, in which two independent genetic loci (S and Z) control self-recognition. S and Z are thought to have arisen from an ancient duplication, common to all grasses. With new chromosome-scale genome data, we examined the genes present at S- and Z-loci, firstly in ryegrass (Lolium perenne), and subsequently in ~20 other grass species. We found that two DUF247 genes and a short unstructured protein (SP/ZP) were present at both S- and Z- in all SI species, while in self-compatible species these genes were often lost or mutated. Expression data suggested that DUF247 genes acted as the male components and SP/ZP were the female components. Consistent with their role in distinguishing self- from non-self, all genes were hypervariable, although key secondary structure features were conserved, including the predicted N-terminal cleavage site of SP/ZP. The evolutionary history of these genes was probed, revealing that specificity groups at the Z-locus arose before the advent of various grass subfamilies/species, while specificity groups at the S-locus arose after the split of Panicoideae, Chloridoideae, Oryzoideae and Pooideae. Finally, we propose a model explaining how the proteins encoded at the S and Z loci might function to specify self-incompatibility.

Predicted Structure and Functions of the Prototypic Alphaherpesvirus Herpes Simplex Virus Type-1 UL37 Tegument Protein.

The alphaherpesvirus UL37 tegument protein is a highly conserved, multi-functional protein. Mutagenesis analysis delineated the UL37 domains necessary for retrograde transport and viral replication. Specifically, the amino-terminal 480 amino acids are dispensable for virus replication in epithelial cell culture, but it is unknown whether this amino-terminal deletion affects UL37 structure and intracellular transport in epithelial cells and neurons. To investigate the structure and function of UL37, we utilized multiple computational approaches to predict and characterize the secondary and tertiary structure and other functional features. The structure of HSV-1 UL37 and Δ481N were deduced using publicly available predictive algorithms. The predicted model of HSV-1 UL37 is a stable, multi-functional, globular monomer, rich in alpha helices, with unfolded regions within the linker and the C-tail domains. The highly flexible C-tail contains predicted binding sites to the dynein intermediate chain, as well as DNA and RNA. Predicted interactions with the cytoplasmic surface of the lipid membrane suggest UL37 is a peripheral membrane protein. The Δ481N truncation did not alter the predicted structure of the UL37 C-terminus protein and its predicted interaction with dynein. We validated these models by examining the replication kinetics and transport of the Δ481N virus toward the nuclei of infected epithelial and neuronal cells. The Δ481N virus had substantial defects in virus spread; however, it exhibited no apparent defects in virus entry and intracellular transport. Using computational analyses, we identified several key features of UL37, particularly the flexible unstructured tail; we then demonstrated that the UL37 C-terminus alone is sufficient to effectively transport the virus towards the nucleus of infected epithelial and neuronal cells.

Evaluation of the Effectiveness of Derived Features of AlphaFold2 on Single-Sequence Protein Binding Site Prediction.

Simple SummaryWith the development of artificial intelligence, researchers can roughly predict the crystal structure of a protein by computer without the need for biological experiments, which provides new ideas and solutions to problems, such as protein-protein interaction and drug-target predictions. In this study, we proposed strategies to combine predicted protein structures with deep learning networks and evaluated them on different protein binding site prediction tasks. Our computational experiment results showed that all proposed strategies could effectively encode structural information for deep learning models.Though AlphaFold2 has attained considerably high precision on protein structure prediction, it is reported that directly inputting coordinates into deep learning networks cannot achieve desirable results on downstream tasks. Thus, how to process and encode the predicted results into effective forms that deep learning models can understand to improve the performance of downstream tasks is worth exploring. In this study, we tested the effects of five processing strategies of coordinates on two single-sequence protein binding site prediction tasks. These five strategies are spatial filtering, the singular value decomposition of a distance map, calculating the secondary structure feature, and the relative accessible surface area feature of proteins. The computational experiment results showed that all strategies were suitable and effective methods to encode structural information for deep learning models. In addition, by performing a case study of a mutated protein, we showed that the spatial filtering strategy could introduce structural changes into HHblits profiles and deep learning networks when protein mutation happens. In sum, this work provides new insight into the downstream tasks of protein-molecule interaction prediction, such as predicting the binding residues of proteins and estimating the effects of mutations.

Hydrolysis improves the inhibition efficacy of bovine lactoferrin against infection by SARS-CoV-2 pseudovirus

The entry of SARS-CoV-2 into host cells may involve the spike protein cleavage by cathepsin L (CTSL). Certain food proteins such as lactoferrin (Lf) inhibit CTSL. The current study investigated the impact of hydrolysis (0–180 min) by proteinase K on electrophoretic pattern, secondary structure, cathepsin inhibitory and SARS-CoV-2 pseudovirus infectivity inhibitory of bovine Lf. Gel electrophoresis indicated that hydrolysis cut Lf molecules to half lobes (∼40 kDa) and produced peptides ≤18 kDa. Approximation of the secondary structural features through analysis of the second-derivative amide I band collected by infra-red spectroscopy suggested a correlative–causative relationship between cathepsin inhibition and the content of helix-unordered structures in Lf hydrolysate. The half maximal inhibitory concentration (IC50) of Lf hydrolysed for 90 min (H90) against CTSL was about 100 times smaller than that of the Lf hydrolysed for 0 min (H0). H90 had also double activity against SARS-CoV-2 pseudo-types infectivity compared with H0.

In silico structural homology modeling and functional characterization of Mycoplasma gallisepticum variable lipoprotein hemagglutin proteins.

Mycoplasma gallisepticum variable lipoprotein hemagglutin (vlhA) proteins are crucial for immune evasion from the host cells, permitting the persistence and survival of the pathogen. However, the exact molecular mechanism behind the immune evasion function is still not clear. In silico physiochemical analysis, domain analysis, subcellular localization, and homology modeling studies have been carried out to predict the structural and functional properties of these proteins. The outcomes of this study provide significant preliminary data for understanding the immune evasion by vlhA proteins. In this study, we have reported the primary, secondary, and tertiary structural characteristics and subcellular localization, presence of the transmembrane helix and signal peptide, and functional characteristics of vlhA proteins from M. gallisepticum strain R low. The results show variation between the structural and functional components of the proteins, signifying the role and diverse molecular mechanisms in functioning of vlhA proteins in host immune evasion. Moreover the 3D structure predicted in this study will pave a way for understanding vlhA protein function and its interaction with other molecules to undergo immune evasion. This study forms the basis for future experimental studies improving our understanding in the molecular mechanisms used by vlhA proteins.