Biophysics Reports

2015 Vol. 1, No. 1

Cover Story

The presence and folding pattern of chromatin iber, typically regarded as the secondary structure of DNA, in eukaryotic cells has been remained elu- sive and controversial. In page 51 of this issue, Li et al prepared ultra-thin- sections of Hela cell with three different ixation and sectioning methods, and analyzed in vivo architecture of chromatin ibers inside Hela nuclei with electron tomography technology. Their results suggested that the chromatin ibers in eukaryotic Hela cells are likely organized in an architecture with a diameter of about 30 nm.

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Biophysics Reports: focus on theoretical and technical advances

Tao Xu

2015, 1(1): 1-1. doi: 10.1007/s41048-015-0010-3

Abstract(125)

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Physics-based RNA structure prediction

Xiaojun Xu, Shi-Jie Chen

2015, 1(1): 2-13. doi: 10.1007/s41048-015-0001-4

Abstract(303)

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Abstract:
Despite the success of RNA secondary structure prediction for simple, short RNAs, the problem of predicting RNAs with long-range tertiary folds remains. Furthermore, RNA 3D structure prediction is hampered by the lack of the knowledge about the tertiary contacts and their thermodynamic parameters. Low-resolution structural modeling enables us to estimate the conformational entropies for a number of tertiary folds through rigorous statistical mechanical calculations. The models lead to 3D tertiary folds at coarse-grained level. The coarse-grained structures serve as the initial structures for all-atom molecular dynamics refinement to build the final all-atom 3D structures. In this paper, we present an overview of RNA computational models for secondary and tertiary structures' predictions and then focus on a recently developed RNA statistical mechanical model—the Vfold model. The main emphasis is placed on the physics behind the models, including the treatment of the non-canonical interactions in secondary and tertiary structure modelings, and the correlations to RNA functions.

Detecting peroxiredoxin hyperoxidation by one-dimensional isoelectric focusing

Zhenbo Cao, Neil J. Bulleid

2015, 1(1): 14-17. doi: 10.1007/s41048-015-0007-y

Abstract(269)

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Abstract:
The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation.

Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification

Lawrence A. Potempa, Zhen-Yu Yao, Shang-Rong Ji, János G. Filep, Yi Wu

2015, 1(1): 18-33. doi: 10.1007/s41048-015-0003-2

Abstract(407)

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Abstract:
The precise function of C-reactive protein (CRP) as a regulator of inflammation in health and disease continues to evolve. The true understanding of its role in host defense responses has been hampered by numerous reports of comparable systems with contradictory interpretations of CRP as a stimulator, suppressor, or benign contributor to such processes. These discrepancies may be explained in part by the existence of a naturally occurring CRP isoform, termed modified CRP (i.e., mCRP), that is expressed when CRP subunits are dissociated into monomeric structures. The free mCRP subunit undergoes a non-proteolytic conformational change that has unique solubility, antigenicity, and bioactivity compared to the subunits that remain associated in the native, pentameric CRP molecule (i.e., pCRP). As specific reagents have been developed to identify and quantify mCRP, it has become apparent that this isoform can be formed spontaneously in calcium-free solutions. Furthermore, mCRP can be expressed on perturbed cell membranes with as little as 24-48 h incubation in tissue culture. Because mCRP has the same size as pCRP subunits as evaluated by SDS-PAGE, its presence in a pCRP reagent would not be apparent using this technique to evaluate purity. Finally, because many antibody reagents purported to be specific for "CRP" contains some, or substantial specificity tomCRP, antigen-detection techniques using such reagents may fail to distinguish the specific CRP isoform detected. All these caveats concerning CRP structures and measurements suggest that the aforementioned contradictory studies may reflect to some extent on distinctive bioactivities of mCRP rather than on pCRP. To provide a reliable, abundant supply of mCRP for separate and comparable studies, a recombinant proteinwas engineered and expressed in E. coli (i.e., recombinant mCRP or rmCRP). Synthesized protein was produced as inclusion bodies which proved difficult to solubilize for purification and characterization. Herein, we describe a method using anhydride reagents to effectively solubilize rmCRP and allow for chromatographic purification in high yield and free of contaminating endotoxin. Furthermore, the purified rmCRP reagent represents an excellent comparable protein to the biologically produced mCRP and as a distinctive reagent from pCRP. Deciphering the true function of CRP in both health and disease requires a knowledge, understanding, and reliable supply of each of its structures so to define the distinctive effects of each on the body's response to tissue damaging events.

An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells

Yan Chen, Zhiping Xia, Lifen Wang, Yong Yu, Pingsheng Liu, Eli Song, Tao Xu

2015, 1(1): 34-40. doi: 10.1007/s41048-015-0008-x

Abstract(652)

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Abstract:
Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.

Phosphorylation and function of DGAT1 in skeletal muscle cells

Jinhai Yu, Yiran Li, Fei Zou, Shimeng Xu, Pingsheng Liu

2015, 1(1): 41-50. doi: 10.1007/s41048-015-0004-1

Abstract(157)

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Abstract:
Aberrant intramuscular triacylglycerol (TAG) storage in human skeletal muscle is closely related to insulin insensitivity. Excessive lipid storage can induce insulin resistance of skeletal muscle, and under severe conditions, lead to type 2 diabetes. The balance of interconversion between diacylglycerol and TAG greatly influences lipid storage and utilization. Diacylglycerol O-acyltransferase 1 (DGAT1) plays a key role in this process, but its activation and phosphorylation requires further dissection. In this study, 12 putative conserved phosphorylation sites of DGAT1 were identified by examining amino acid conservation of DGAT1 in different species. Another 12 putative phosphorylation sites were also found based on bioinformatics predictions and previous reports. Meanwhile, several phosphorylation sites of DGAT1 were verified by phosphorylation mass spectrometry analysis of purified DGAT1 from mouse myoblast C2C12 cells. Using single point mutations, a regulatory role of 3 putative phosphorylation sites was dissected. Finally, using truncation mutations, a potential domain of DGAT1 that was involved in the regulation of enzymatic activity was revealed. This study provides useful information for further understanding DGAT1 activity regulation.

Analysis of chromatin bersin Hela cells with electron tomography

Xiaomin Li, Hongli Feng, Jianguo Zhang, Lei Sun, Ping Zhu

2015, 1(1): 51-60. doi: 10.1007/x41048-015-0009-9

Abstract(225)

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Abstract:
The presence and folding pattern of chromatin in eukaryotic cells remain elusive and controversial. In this study, we prepared ultra-thin sections of Hela cells with three different fixation and sectioning methods, i.e., chemical fixation, high pressure freezing with freeze substitution, and cryo-ultramicro- tomy with SEM-FIB (focused ion beam), and analyzed in vivo architecture of chromatin fibers in Hela nuclei with electron tomography technology. The results suggest that the chromatin fibers in eukaryotic Hela cells are likely organized in an architecture with a diameter of about 30 nm.