Template-based protein structure modeling using the RaptorX web server

A key challenge of modern biology is to uncover the functional role of the protein entities that compose cellular proteomes. To this end, the availability of reliable three-dimensional atomic models of proteins is often crucial. This protocol presents a community-wide web-based method using RaptorX (http://raptorx.uchicago.edu/) for protein secondary structure prediction, template-based tertiary structure modeling, alignment quality assessment and sophisticated probabilistic alignment sampling. RaptorX distinguishes itself from other servers by the quality of the alignment between a target sequence and one or multiple distantly related template proteins (especially those with sparse sequence profiles) and by a novel nonlinear scoring function and a probabilistic-consistency algorithm. Consequently, RaptorX delivers high-quality structural models for many targets with only remote templates. At present, it takes RaptorX ~35 min to finish processing a sequence of 200 amino acids. Since its official release in August 2011, RaptorX has processed ~6,000 sequences submitted by ~1,600 users from around the world.     

Identification of proteins interacting with Arabidopsis ACD11

The Arabidopsis ACD11 gene encodes a sphingosine transfer protein and was identified by the accelerated cell death phenotype of the loss of function acd11 mutant, which exhibits heightened expression of genes involved in the disease resistance hypersensitive response (HR). We used ACD11 as bait in a yeast two-hybrid screen of an Arabidopsis cDNA library to identify ACD11 interacting proteins. One interactor identified is a protein of unknown function with an RNA recognition motif (RRM) designated BPA1 (binding partner of ACD11). Co-immunoprecipitation experiments confirmed the ACD11-BPA1 interactions in vivo and in vitro. Two other ACD11 interactors (PRA7 and PRA8) are homologous to each other and to mammalian PRA1, and both were subsequently shown to interact with BPA1 in yeast. A fourth interactor (VAP27-1) is homologous to mammalian VAP-A, and was found to interact more strongly with a homolog of ACD11 than ACD11 itself. All interactors were shown to be associated with membrane fractions, suggesting that ACD11 function could be related to the regulation of membrane compartments.     

Reversible dimerization of acid-denatured ACBP controlled by helix A4

The peptide segment corresponding to helix A4 in acyl-coenzyme-A-binding protein (ACBP) is an exceptionally stable helix in the denatured state of the protein as well as in its isolated form. Circular dichroism spectroscopy showed an alpha-helix content in the helix A4 peptide (HA4) of 45%, and under denaturing conditions at pH 2.3, helix conformations are still populated in 24% of the ensemble of molecules. The structure of HA4 at atomic resolution was assessed using nuclear magnetic resonance (NMR) spectroscopy. Long-range NOEs between remote residues at opposite peptide ends suggested the formation of an antiparallel homodimer, and the resulting structure was treated as the minimum higher-order structure. The dimerization property of helix A4 is maintained in the full-length protein under denaturing conditions. NMR diffusion studies and concentration-dependent experiments on ACBP at low pH proved the formation of dimers and revealed a cooperative stabilization of helix A4 in this process. This emphasizes its special role in the structure formation in the denatured state of ACBP. No dimers are formed in the presence of guanidine hydrochloride, which underlines the fundamental difference between the nature of these two denatured states.     

NMR and interval PLS as reliable methods for determination of cholesterol in rodent lipoprotein fractions

Risk of cardiovascular disease is related to cholesterol distribution in different lipoprotein fractions. Lipoproteins in rodent model studies can only reliably be measured by time- and plasma-consuming fractionation. An alternative method to measure cholesterol distribution in the lipoprotein fractions in rat plasma is presented in this paper. Plasma from two rat studies (n = 68) was used in determining the lipoprotein profile by an established ultracentrifugation method and proton nuclear magnetic resonance (NMR) spectra of replicate samples was obtained. From the ultracentrifugation reference data and the NMR spectra, an interval partial least-square (iPLS) regression model to predict the amount of cholesterol in the different lipoprotein fractions was developed. The relative errors of the prediction models were between 12 and 33% and had correlation coefficients (r) between 0.96 and 0.84. The models were tested with an independent test set giving prediction errors between 19 and 46% and r between 0.96 and 0.76. Prediction of High, Low and Very Low Density Lipoprotein (HDL, LDL and VLDL) and total cholesterol was conducted in a study where rats had been supplemented with two doses of air-dried apple-powder. No significant difference in LDL, VLDL and total cholesterol was observed between the groups. The high apple-powder (20%) group had significantly lower HDL cholesterol (11%, P = 0.0452) than the control group. It is concluded that the iPLS approach yielded excellent regression models and thus univocal established chemometric analysis of NMR spectra of rat plasma as a strong and efficient way to quantify lipoprotein fractions in rat studies.     

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (⁰) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.     

Micro‐ and macroheterogeneity of N‐glycosylation yields size and charge isoforms of human sex hormone binding globulin circulating in serum

Human sex hormone binding globulin (hSHBG) is a serum glycoprotein central to the transport and targeted delivery of sex hormones to steroid‐sensitive tissues. Several molecular mechanisms of action of hSHBG, including the function of its attached glycans remain unknown. Here, we perform a detailed site‐specific characterization of the N‐ and O‐linked glycosylation of serum‐derived hSHBG. MS‐driven glycoproteomics and glycomics combined with exoglycosidase treatment were used in a bottom‐up and top‐down manner to determine glycosylation sites, site‐specific occupancies and monosaccharide compositions, detailed glycan structures, and the higher level arrangement of glycans on intact hSHBG. It was found that serum‐derived hSHBG is N‐glycosylated at Asn³⁵¹ and Asn³⁶⁷ with average molar occupancies of 85.1 and 95.3%, respectively. Both sites are occupied by the same six sialylated and partly core fucosylated bi‐ and triantennary N‐Glycoforms with lactosamine‐type antennas of the form (±NeuAcα6)Galβ4GlcNAc. N‐Glycoforms of Asn³⁶⁷ were slightly more branched and core fucosylated than Asn³⁵¹ N‐glycoforms due probably to a more surface‐exposed glycosylation site. The N‐terminal Thr⁷ was fully occupied by the two O‐linked glycans NeuAcα3Galβ3(NeuAcα6)GalNAc (where NeuAc is N‐acetylneuraminic acid and GalNAc is N‐acetylgalactosamine) and NeuAcα3Galβ3GalNAc in a 1:6 molar ratio. Electrophoretic analysis of intact hSHBG revealed size and charge heterogeneity of the isoforms circulating in blood serum. Interestingly, the size and charge heterogeneity were shown to originate predominantly from differential Asn³⁵¹ glycan occupancies and N‐glycan sialylation that may modulate the hSHBG activity. To date, this work represents the most detailed structural map of the heterogeneous hSHBG glycosylation, which is a prerequisite for investigating the functional aspects of the hSHBG glycans.