An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide

Guan, Yihong; Zhu, Qinfang; Huang, Delai; Zhao, Shanshan; Lo, Li Jan; Peng, Jinrong

doi:10.1038/srep13370

Cited by 118 publications

(87 citation statements)

References 35 publications

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“…3A; three replicate samples are presented). Interestingly, we see a shift in the apparent molecular mass of Cx45 6E compared to the WT; a result consistent with a study that examined the effects of proteins containing disproportionate numbers of acidic amino acids [32]. We also screened for Cx43 expression as its residual expression could confound results in subsequent studies due to presence of Cx43 homomeric or Cx43/Cx45 heteromeric channels.…”

Section: Resultssupporting

confidence: 86%

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

Trease

Capuccino

Contreras

et al. 2017

Journal of Molecular and Cellular Cardiology

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Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD ~100 nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.

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Section: Resultssupporting

confidence: 86%

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

Trease

Capuccino

Contreras

et al. 2017

Journal of Molecular and Cellular Cardiology

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“…Consequently, observed migration bands of acidic proteins in SDS-PAGE gels are well known to not correspond directly to their molecular weight (MW). Fortunately, a carefully devised correction equation was recently shown to address this long standing problem [37]. Our main line of argumentation relies on the observation of protein isoforms with sizes (inferred by MW) quantitatively consistent with translation products from intraRNAs.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore it is assumed implicitly that these proteins had their N-terminal methionine cleaved and the mature form was recorded in the database, not being a degradative proteolysis event. All validation cases based on protein gels were acidity-corrected [37] implicitly assuming that the equation is universally applicable and grounded on the fact that the known full-length protein MWs were properly adjusted. Finally, the current generation sequencing platforms allows, even with the paired-end improvement, a limited coverage of RNA lengths.…”

Section: Discussionmentioning

confidence: 99%

“…Apparent MW due to differential migration of acidic protein was calculated by the equations M c = M + ∆ M ( M, s ) and ∆ M ( M, s ) = n ( s ) · (276.5· k ( s )/ n ( s ) – 31.33), where: M is the predicted protein MW; M c is the apparent MW after correction; n ( s ) is the number of amino acid residues and k ( s ) is the number of glutamic acid (E) or aspartic acid (D) amino acid residues in the protein sequence s [37]. However, ∆ M ( M, s ) = 0 if k ( s )/ n ( s ) < 0.114 or if k ( s )/ n ( s ) > 0.511, i.e ., there is no correction.…”

Section: Methodsmentioning

confidence: 99%

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Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

et al. 2018

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Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.

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“…The first reason could be the binding of proteins to SDS leading to formation of SDS-complexes and resulting in modification and oligomerization of proteins [22]. Other reasons could be post translational modifications like glycosylation, ubiquitination or phosphorylation etc., presence of acidic amino acids in the protein [23], incomplete unfolding of proteins, or presence of proline-rich regions which lend rigidity to the protein backbone. Lower observed molecular weight of some proteins could be due to truncation of proteins as some proteins carry protease sensitive motifs and may get cleaved off.…”

Section: Discussionmentioning

confidence: 99%

Proteomic Investigation of Photorhabdus Bacteria for Nematode-Host Specificity

et al. 2016

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Majority of animals form symbiotic relationships with bacteria. Based on the number of bacterial species associating with an animal, these symbiotic associations can be mono-specific, relatively simple (2-25 bacterial species/ animal) or highly complex ([10 2 -10 3 bacterial species/animal). Photorhabdus (family-Enterobacteriaceae) forms a mono-specific symbiotic relationship with the entomopathogenic nematode Heterorhabditis. This system provides a tractable genetic model for animal-microbe symbiosis studies. Here, we investigated the bacterial factors that may be responsible for governing host specificity between nematode and their symbiont bacteria using proteomics approach. Total protein profiles of P. luminescens ssp. laumondii (host nematode-H. bacteriophora) and P. luminescens ssp. akhurstii (host nematode-H. indica) were compared using 2-D gel electrophoresis, followed by identification of differentially expressed proteins by MALDI-TOF MS. Thirty-nine unique protein spots were identified -24 from P. luminescens ssp. laumondii and 15 from P. luminescens ssp. akhurstii. These included proteins that might be involved in determining host specificity directly (for e.g. pilin FimA, outer membrane protein A), indirectly through effect on bacterial secondary metabolism (for e.g. malate dehydrogenase Mdh, Pyruvate formate-lyase PflA, flavo protein WrbA), or in a yet unknown manner (for e.g. hypothetical proteins, transcription regulators). Further functional validation is needed to establish the role of these bacterial proteins in nematode-host specificity.

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An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide

Cited by 118 publications

References 35 publications

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

Proteomic Investigation of Photorhabdus Bacteria for Nematode-Host Specificity

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