Mutagenesis Objective Search and Selection Tool (MOSST): an algorithm to predict structure-function related mutations in proteins

<p>Abstract</p> <p>Background</p> <p>Functionally relevant artificial or natural mutations are difficult to assess or predict if no structure-function information is available for a protein. This is especially important to correctly identify functionally significant non-synonymous single nucleotide polymorphisms (nsSNPs) or to design a site-directed mutagenesis strategy for a target protein. A new and powerful methodology is proposed to guide these two decision strategies, based only on conservation rules of physicochemical properties of amino acids extracted from a multiple alignment of a protein family where the target protein belongs, with no need of explicit structure-function relationships.</p> <p>Results</p> <p>A statistical analysis is performed over each amino acid position in the multiple protein alignment, based on different amino acid physical or chemical characteristics, including hydrophobicity, side-chain volume, charge and protein conformational parameters. The variances of each of these properties at each position are combined to obtain a global statistical indicator of the conservation degree of each property. Different types of physicochemical conservation are defined to characterize relevant and irrelevant positions. The differences between statistical variances are taken together as the basis of hypothesis tests at each position to search for functionally significant mutable sites and to identify specific mutagenesis targets. The outcome is used to statistically predict physicochemical consensus sequences based on different properties and to calculate the amino acid propensities at each position in a given protein. Hence, amino acid positions are identified that are putatively responsible for function, specificity, stability or binding interactions in a family of proteins. Once these key functional positions are identified, position-specific statistical distributions are applied to divide the 20 common protein amino acids in each position of the protein's primary sequence into a group of functionally non-disruptive amino acids and a second group of functionally deleterious amino acids.</p> <p>Conclusions</p> <p>With this approach, not only conserved amino acid positions in a protein family can be labeled as functionally relevant, but also non-conserved amino acid positions can be identified to have a physicochemically meaningful functional effect. These results become a discriminative tool in the selection and elaboration of rational mutagenesis strategies for the protein. They can also be used to predict if a given nsSNP, identified, for instance, in a genomic-scale analysis, can have a functional implication for a particular protein and which nsSNPs are most likely to be functionally silent for a protein. This analytical tool could be used to rapidly and automatically discard any irrelevant nsSNP and guide the research focus toward functionally significant mutations. Based on preliminary results and applications, this technique shows promising performance as a valuable bioinformatics tool to aid in the development of new protein variants and in the understanding of function-structure relationships in proteins.</p

MicroRNA-96 Directly Inhibits γ-Globin Expression in Human Erythropoiesis

Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (α2β2), becomes predominant. Several studies have indicated that expression of the HbF subunit γ-globin might be regulated post-transcriptionally. This could be confered by ∼22-nucleotide long microRNAs that associate with argonaute proteins to specifically target γ-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that γ-globin mRNA was associated with argonaute 2 isolated from reticulocytes that contain low levels of HbF (<1%), whereas association was significantly lower in reticulocytes with high levels of HbF (90%). Comparing microRNA expression in reticulocytes from cord blood and adult blood, we identified several miRNAs that were preferentially expressed in adults, among them miRNA-96. The overexpression of microRNA-96 in human ex vivo erythropoiesis decreased γ-globin expression by 50%, whereas the knock-down of endogenous microRNA-96 increased γ-globin expression by 20%. Moreover, luciferase reporter assays showed that microRNA-96 negatively regulates expression of γ-globin in HEK293 cells, which depends on a seedless but highly complementary target site located within the coding sequence of γ-globin. Based on these results we conclude that microRNA-96 directly suppresses γ-globin expression and thus contributes to HbF regulation

Nature of protein family signatures: Insights from singular value analysis of position-specific scoring matrices

Position-specific scoring matrices (PSSMs) are useful for detecting weak homology in protein sequence analysis, and they are thought to contain some essential signatures of the protein families. In order to elucidate what kind of ingredients constitute such family-specific signatures, we apply singular value decomposition to a set of PSSMs and examine the properties of dominant right and left singular vectors. The first right singular vectors were correlated with various amino acid indices including relative mutability, amino acid composition in protein interior, hydropathy, or turn propensity, depending on proteins. A significant correlation between the first left singular vector and a measure of site conservation was observed. It is shown that the contribution of the first singular component to the PSSMs act to disfavor potentially but falsely functionally important residues at conserved sites. The second right singular vectors were highly correlated with hydrophobicity scales, and the corresponding left singular vectors with contact numbers of protein structures. It is suggested that sequence alignment with a PSSM is essentially equivalent to threading supplemented with functional information. The presented method may be used to separate functionally important sites from structurally important ones, and thus it may be a useful tool for predicting protein functions.Comment: 22 pages, 7 figures, 4 table

A versatile modelling approach to determine the hydrophobicity of peptides at the atomic level

This study describes a versatile computational method to determine the hydrophobicity of small peptides at the atomic level. Free energies of transfer for individual atoms in peptide structures were derived, utilising two specifically defined parameters: (i) the water-excluding distance to define the dynamic interface between a peptide solute and its surrounding solvent and (ii) the corresponding hydrophobicity index as a relative measure for water occlusion/repulsion. The method was tested on a range of small peptide models (Ac-X-NH2, G-X-G, Ac-WL-X-LL and Ac-GG-X-GG-NH2) and several derivatives of these structures, whereby X was any of the 20 most common amino acids that naturally occur in polypeptides or proteins. The advantage of this new method lies in its versatility, ease to implement and capability to provide information on the hydrophobicity characteristics at the atomic level. The approach also encapsulates the impact of factors that influence these properties, but which have hitherto been difficult to accurately quantify, e.g. steric hindrance or proximity effects due to nearby polarised atoms. The method is not conditional on the knowledge of hydrophobicity parameters from the literature and does not require a sophisticated computer software/hardware to enable the atomic solvent-accessible surface areas or other hydrophobicity parameters to be de novo obtained.</p

Tuning Activity of Antimicrobial Peptides by Lipidation

Antimicrobial peptides (AMPs) are amino acid-based bioactive molecules that specifically target microbes. As such, they are a potent class of antibiotics, especially against bacterial infections. Naturally occurring AMPs are usually too long to be considered for therapeutic applications. To solve this, short sequences that mimic the activity of AMPs are designed. However, such endeavors are often accompanied with a reduction in antibacterial activity. To counter this, lipophilic molecules can be attached that function as a lipid anchor and target the short sequence to the bacterial membrane. For a range of short AMPs, this strategy has proven to lead to more active constructs. Although these lipidated short AMPs often work as complex target specific surfactants, more delicate modes of action that do not deviate too much from the nonlipidated counterparts are also known. This is readily observed by the large differences in activities that are detected when alterations in the lipid chain length and chirality of the amino acids residues are implemented. It is not uncommon to see that inactive or poorly active short AMPs can be turned into potent antibacterial agents. Importantly, selectivity of the short lipidated AMPs (lipoAMPs) for the bacterial membrane can be enhanced by alteration of the amino acid chirality. This strategy has led to lipoAMPs with submicromolar activities; in fact, activities that rival that of vancomycin have been observed for several short AMPs. Future research needs to determine (i) the effect of lipidation on the formation of lipid rafts in the bacterial membrane, (ii) if structural complications like branched lipids or chiral substituents on the lipid chain can be used to further increase the activity and selectivity of the conjugates, and (iii) if additional functionalities other than a membrane-anchoring ability can be bestowed on the lipid chain, e.g., redox activity or scavenger for small molecular components that traverse the lipid membrane. The interplay between degree of lipophilicity and the chirality of the amino acids of the AMP also needs further exploration, especially to see if more potent and selective (lipo)AMPs can be obtained that can be applied systemically. It may also be advisable to measure the most potent lipoAMPs in a centralized facility in order to obtain objective and comparable antibacterial activities