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Virus Isoelectric Point Estimation: Theories and Methods.

ABSTRACT: Much of virus fate, both in the environment and in physical/chemical treatment, is dependent on electrostatic interactions. Developing an accurate means of predicting virion isoelectric point (pI) would help to understand and anticipate virus fate and transport, especially for viruses that are not readily propagated in the lab. One simple approach to predicting pI estimates the pH at which the sum of charges from ionizable amino acids in capsid proteins approaches zero. However, predicted pIs based on capsid charges frequently deviate by several pH units from empirically measured pIs. Recently, the discrepancy between empirical and predicted pI was attributed to the electrostatic neutralization of predictable polynucleotide-binding regions (PBRs) of the capsid interior. In this paper, we review models presupposing (i) the influence of the viral polynucleotide on surface charge or (ii) the contribution of only exterior residues to surface charge. We then compare these models to the approach of excluding only PBRs and hypothesize a conceptual electrostatic model that aligns with this approach. The PBR exclusion method outperformed methods based on three-dimensional (3D) structure and accounted for major discrepancies in predicted pIs without adversely affecting pI prediction for a diverse range of viruses. In addition, the PBR exclusion method was determined to be the best available method for predicting virus pI, since (i) PBRs are predicted independently of the impact on pI, (ii) PBR prediction relies on proteome sequences rather than detailed structural models, and (iii) PBR exclusion was successfully demonstrated on a diverse set of viruses. These models apply to nonenveloped viruses only. A similar model for enveloped viruses is complicated by a lack of data on enveloped virus pI, as well as uncertainties regarding the influence of the phospholipid envelope on charge and ion gradients.

SUBMITTER: Heffron J

PROVIDER: S-EPMC7848896 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In any macromolecular polyprotic system-for example protein, DNA or RNA-the isoelectric point-commonly referred to as the pI-can be defined as the point of singularity in a titration curve, corresponding to the solution pH value at which the net overall surface charge-and thus the electrophoretic mobility-of the ampholyte sums to zero. Different modern analytical biochemistry and proteomics methods depend on the isoelectric point as a principal feature for protein and peptide characterization. Protein separation by isoelectric point is a critical part of 2-D gel electrophoresis, a key precursor of proteomics, where discrete spots can be digested in-gel, and proteins subsequently identified by analytical mass spectrometry. Peptide fractionation according to their pI is also widely used in current proteomics sample preparation procedures previous to the LC-MS/MS analysis. Therefore accurate theoretical prediction of pI would expedite such analysis. While such pI calculation is widely used, it remains largely untested, motivating our efforts to benchmark pI prediction methods.Using data from the database PIP-DB and one publically available dataset as our reference gold standard, we have undertaken the benchmarking of pI calculation methods. We find that methods vary in their accuracy and are highly sensitive to the choice of basis set. The machine-learning algorithms, especially the SVM-based algorithm, showed a superior performance when studying peptide mixtures. In general, learning-based pI prediction methods (such as Cofactor, SVM and Branca) require a large training dataset and their resulting performance will strongly depend of the quality of that data. In contrast with Iterative methods, machine-learning algorithms have the advantage of being able to add new features to improve the accuracy of prediction.yperez@ebi.ac.ukThe software and data are freely available at https://github.com/ypriverol/pIRSupplementary information: Supplementary data are available at Bioinformatics online.

Project description:We previously described the photoactivated depot or PAD approach that allows for the light control of therapeutic protein release. This approach relies on the ability to use light to change a protein's solubility. Traditionally this was accomplished by linking the protein to an insoluble but injectable polymer via a light cleaved linker. This allows the injected material to remain at the site of injection, until transcutaneous irradiation breaks the link between polymer and protein, permitting the protein to be absorbed. However, there are multiple problems associated with polymer based approaches: The polymer makes up a majority of the material, making it inefficient. In addition, after protein release, the polymer has to be cleared from the body, a significant design challenge. In this work, we create materials that form photoactivated depots of insulin without the need for polymers, by linking photolysis to an isoelectric point shift, which itself is linked to a solubility shift. Specifically, we linked basic groups to insulin via a light cleaved linker. These shift the normal pI of insulin from 5.4 to approximately 7. The result of this incorporation are materials that are completely soluble in mildly acidic solutions but precipitate upon injection into a pH 7 environment, i.e., the skin. We successfully synthesized four such modified insulins, demonstrating that their pI values were shifted in the expected manner. We then analyzed one of them, P2-insulin, in detail, demonstrating that it behaves as designed: It is soluble in a formulation pH of 4, but precipitates at pH 7.2, its approximate pI value. Upon irradiation, the photocleavable link to insulin is broken, and completely native and soluble insulin is released from the depot in a well behaved, first order fashion. These materials are 90% therapeutic, form completely soluble and injectable formulations in mildly acidic conditions, form insoluble depots at neutral pH, efficiently release soluble protein from these depots when irradiated, and leave behind only small easily absorbed molecules after irradiation. As such they approach ideality for photoactivated depot materials.

Project description:BACKGROUND:Cell contain diverse array of proteins with different molecular weight and isoelectric point (pI). The molecular weight and pI of protein play important role in determining the molecular biochemical function. Therefore, it was important to understand the detail regarding the molecular weight and pI of the plant proteins. RESULTS:A proteome-wide analysis of plant proteomes from 145 species revealed a pI range of 1.99 (epsin) to 13.96 (hypothetical protein). The spectrum of molecular mass of the plant proteins varied from 0.54 to 2236.8?kDa. A putative Type-I polyketide synthase (22244 amino acids) in Volvox carteri was found to be the largest protein in the plant kingdom. However, Type-I polyketide synthase was not found in higher plant species. Titin (806.46?kDa) and misin/midasin (730.02?kDa) were the largest proteins identified in higher plant species. The pI and molecular weight of the plant proteins showed a trimodal distribution. An acidic pI (56.44% of proteins) was found to be predominant over a basic pI (43.34% of proteins) and the abundance of acidic pI proteins was higher in unicellular algae species relative to multicellular higher plants. In contrast, the seaweed, Porphyra umbilicalis, possesses a higher proportion of basic pI proteins (70.09%). Plant proteomes were also found to contain selenocysteine (Sec), amino acid that was found only in lower eukaryotic aquatic plant lineage. Amino acid composition analysis showed Leu was high and Trp was low abundant amino acids in the plant proteome. Additionally, the plant proteomes also possess ambiguous amino acids Xaa (unknown), Asx (asparagine or aspartic acid), Glx (glutamine or glutamic acid), and Xle (leucine or isoleucine) as well. CONCLUSION:The diverse molecular weight and isoelectric point range of plant proteome will be helpful to understand their biochemical and functional aspects. The presence of selenocysteine proteins in lower eukaryotic organism is of interest and their expression in higher plant system can help us to understand their functional role.

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Virus Isoelectric Point Estimation: Theories and Methods.

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