scholarly journals Inline Liquid Chromatography-Fast Photochemical Oxidation of Proteins Allows for Targeted Structural Analysis of Conformationally Heterogeneous Mixtures

2019 ◽  
Author(s):  
Surendar Tadi ◽  
Sandeep K. Misra ◽  
Joshua S. Sharp
Keyword(s):  
Structural Analysis ◽  
Heat Shock ◽  
Weighted Average ◽  
Difficult Problem ◽  
Photochemical Oxidation ◽  
Size Exclusion ◽  
Accurate Information ◽  
Aggregation Process ◽  
Hydroxyl Radical Protein Footprinting ◽  
Exclusion Chromatography

AbstractStructural analysis of proteins in a conformationally heterogeneous mixture has long been a difficult problem in structural biology. In structural analysis by covalent labeling mass spectrometry, conformational heterogeneity results in data reflecting a weighted average of all conformers, complicating data analysis and potentially causing misinterpretation of results. Here, we describe a method coupling size exclusion chromatography (SEC) with hydroxyl radical protein footprinting using inline fast photochemical oxidation of proteins (FPOP). Using a controlled synthetic mixture of holomyoglobin and apomyoglobin, we demonstrate that we can achieve accurate footprints of each conformer using LC-FPOP when compared to off-line FPOP of each pure conformer. We then applied LC-FPOP to analyze the adalimumab heat-shock aggregation process. We found that the LC-FPOP footprint of unaggregated adalimumab was consistent with a previously published footprint of the native IgG. The LC-FPOP footprint of the aggregation product indicated that heat shock aggregation primarily protected the hinge region, suggesting this region is involved with the heat shock aggregation process of this molecule. LC-FPOP offers a new method to probe dynamic conformationally heterogeneous mixtures such as biopharmaceutical aggregates, and obtain accurate information on the topography of each conformer.

scholarly journals Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis

Open Biology ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 190258
Author(s):  
K. C. T. Riciluca ◽  
A. C. Borges ◽  
J. F. R. Mello ◽  
U. C. de Oliveira ◽  
D. C. Serdan ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Signal Peptide ◽  
Oxygen Transport ◽  
Three Dimensional ◽  
Size Exclusion ◽  
Tem Analysis ◽  
Arthropod Species ◽  
Dimensional Reconstruction ◽  
Exclusion Chromatography ◽  
Respiratory Proteins

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

scholarly journals Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp)

2021 ◽  
Vol 22 (15) ◽  
pp. 7777
Author(s):  
Lydia K. Muranova ◽  
Vladislav M. Shatov ◽  
Andrey V. Slushchev ◽  
Nikolai B. Gusev
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Quaternary Structure ◽  
Small Heat Shock Protein ◽  
Apparent Molecular Weight ◽  
Size Exclusion ◽  
Wild Type ◽  
Simple Method ◽  
Exclusion Chromatography ◽  
Shock Proteins

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17–29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the β7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

scholarly journals Replacement of Arg in the conserved N-terminal RLFDQxFG motif affects physico-chemical properties and chaperone-like activity of human small heat shock protein HspB8 (Hsp22)

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253432
Author(s):  
Vladislav M. Shatov ◽  
Nikolai N. Sluchanko ◽  
Nikolai B. Gusev
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Chemical Properties ◽  
Small Heat Shock Protein ◽  
Size Exclusion ◽  
Terminal Domain ◽  
Physico Chemical ◽  
Exclusion Chromatography ◽  
Erk1 Protein

The small heat shock protein (sHsp) called HspB8 (formerly, Hsp22) is one of the least typical sHsp members, whose oligomerization status remains debatable. Here we analyze the effect of mutations in a highly conservative sequence located in the N-terminal domain of human HspB8 on its physico-chemical properties and chaperone-like activity. According to size-exclusion chromatography coupled to multi-angle light scattering, the wild type (WT) HspB8 is present as dominating monomeric species (~24 kDa) and a small fraction of oligomers (~60 kDa). The R29A amino acid substitution leads to the predominant formation of 60-kDa oligomers, leaving only a small fraction of monomers. Deletion of the 28–32 pentapeptide (Δ mutant) results in the formation of minor quantities of dimers (~49 kDa) and large quantities of the 24-kDa monomers. Both the WT protein and its Δ mutant efficiently bind a hydrophobic probe bis-ANS and are relatively rapidly hydrolyzed by chymotrypsin, whereas the R29A mutant weakly binds bis-ANS and resists chymotrypsinolysis. In contrast to HspB8 WT and its Δ mutant, which are well phosphorylated by cAMP-dependent and ERK1 protein kinases, the R29A mutant is poorly phosphorylated. R29A mutation affects the chaperone-like activity of HspB8 measured in vitro. It is concluded that the irreplaceable Arg residue located in the only highly conservative motif in the N-terminal domain of all sHsp proteins affects the oligomeric structure and key properties of HspB8.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

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