抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of “gel shifting” has perplexed biochemists and biologists for decades. We identify a mechanism for “gel shifting” that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ∼80–101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80–101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80–101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).

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Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

Protein-surfactant interaction is widely studied to understand stability and structural changes in proteins. In this Article, we have investigated SDS-induced unfolding of RNase A using absorbance, intrinsic fluorescence of the protein, anisotropy, TNS fluorescence, and near- and far-UV circular dichroism. Unfolding titration curves obtained from the absorbance and fluorescence changes were fitted into a five-state protein unfolding model by assuming formation of three intermediate states. Free energy changes and m-values of all four transitions between the native and unfolded state were evaluated. The transitions are categorized into two different regions. Region I, up to 0.5 mM of SDS, involves ionic interaction between the protein and SDS where the secondary and tertiary structure of the protein is altered to a less extent. In region II, hydrophobic interaction dominates and has two distinct transitions. The first transition arises from the aggregation of surfactant molecules around the protein hydrophobic sites. In the following transition, the micelles probably expand more, and a few more hydrophobic sites are occupied by the surfactant. In this region, the tertiary contacts are completely broken, and almost 50% of the secondary structure is lost. The aggregation of SDS around the protein starts well below the CMC. These conformational changes can be explained by the necklace and beads model, and the free energy of formation of such a complex for the RNase A-SDS system is found to be 5.2 (±1.0) kcal mol(-1). The probable interaction sites and the mechanism of unfolding have been discussed in detail.

Protein–Surfactant Interaction: Sodium Dodecyl Sulfate-Induced Unfolding of Ribonuclease A

A previous study, using capillary electrophoresis (CE) [J. Am. Chem. Soc.2008, 130, 17384–17393], reported that six discrete complexes of ubiquitin (UBI) and sodium dodecyl sulfate (SDS) form at different concentrations of SDS along the pathway to unfolding of UBI in solutions of SDS. One complex (which formed between 0.8 and 1.8 mM SDS) consisted of native UBI associated with approximately 11 molecules of SDS. The current study used CE and 15N/13C–1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy to identify residues in folded UBI that associate specifically with SDS at 0.8–1.8 mM SDS, and to correlate these associations with established biophysical and structural properties of this well-characterized protein. The ability of the surface charge and hydrophobicity of folded UBI to affect the association with SDS (at concentrations below the CMC) was studied, using CE, by converting lys-e-NH3+ to lys-e-NHCOCH3 groups. According to CE, the acetylation of lysine residues inhibited the binding ...

http://dash.harvard.edu/bitstream/handle/1/12967831/93963192.pdf?sequence=1

Complexes of Native Ubiquitin and Dodecyl Sulfate Illustrate the Nature of Hydrophobic and Electrostatic Interactions in the Binding of Proteins and Surfactants

Changes in fluorescent dissolved organic matter upon interaction with anionic surfactant as revealed by EEM-PARAFAC and two dimensional correlation spectroscopy.

This paper characterizes the complexes formed by a small protein, ubiquitin (UBI), and a negatively charged surfactant, sodium dodecyl sulfate (SDS), using capillary electrophoresis (CE), circular dichroism (CD), and amide hydrogen-deuterium exchange (HDX; as monitored by mass spectroscopy, MS). Capillary electrophoresis of complexes of UBI and SDS, at apparent equilibrium, at concentrations of SDS ranging from sub-micellar and sub-denaturing to micellar and denaturing, revealed multiple complexes of UBI and SDS of the general composition UBI-SDS(n). Examination of electrophoretic mobilities of complexes of UBI and SDS as a function of the concentration of SDS provided a new way to characterize the interaction of this protein with SDS and established key characteristics of this system: e.g., the reversibility of the formation of the complexes, their approximate chemical compositions, and the pathway of SDS binding to UBI. The work identified, in addition to SDS-saturated UBI, at least six groups of complexes of UBI with SDS, within which four groups were populated with complexes of distinct stoichiometries: UBI-SDS(approximately 11), UBI-SDS(approximately 25), UBI-SDS(approximately 33), and UBI-SDS(approximately 42). CD spectroscopy and amide HDX of the UBI-SDS(n) complexes suggested that many of the UBI-SDS(n) complexes (n > 11) have greater alpha-helical content than native UBI. Capillary electrophoresis provides a level of detail about interactions of proteins and SDS that has not previously been accessible, and CE is an analytical and biophysical method for studies of interactions of proteins and surfactants that is both convenient and practical. This study sheds light on the formation of the enigmatic protein-SDS complexes formed during SDS polyacrylamide gel electrophoresis and brings a new tool to the study of proteins and detergents.

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Emanuel Carillho

Papers

Pathway for unfolding of ubiquitin in sodium dodecyl sulfate, studied by capillary electrophoresis.