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

Unlike other methods for docking ligands to the rigid 3D structure of a known protein receptor, Glide approximates a complete systematic search of the conformational, orientational, and positional space of the docked ligand In this search, an initial rough positioning and scoring phase that dramatically narrows the search space is followed by torsionally flexible energy optimization on an OPLS-AA nonbonded potential grid for a few hundred surviving candidate poses The very best candidates are further refined via a Monte Carlo sampling of pose conformation; in some cases, this is crucial to obtaining an accurate docked pose Selection of the best docked pose uses a model energy function that combines empirical and force-field-based terms Docking accuracy is assessed by redocking ligands from 282 cocrystallized PDB complexes starting from conformationally optimized ligand geometries that bear no memory of the correctly docked pose Errors in geometry for the top-ranked pose are less than 1 A in nearly ha

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Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy.

Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes

A novel scoring function to estimate protein-ligand binding affinities has been developed and implemented as the Glide 4.0 XP scoring function and docking protocol. In addition to unique water desolvation energy terms, protein-ligand structural motifs leading to enhanced binding affinity are included: (1) hydrophobic enclosure where groups of lipophilic ligand atoms are enclosed on opposite faces by lipophilic protein atoms, (2) neutral-neutral single or correlated hydrogen bonds in a hydrophobically enclosed environment, and (3) five categories of charged-charged hydrogen bonds. The XP scoring function and docking protocol have been developed to reproduce experimental binding affinities for a set of 198 complexes (RMSDs of 2.26 and 1.73 kcal/mol over all and well-docked ligands, respectively) and to yield quality enrichments for a set of fifteen screens of pharmaceutical importance. Enrichment results demonstrate the importance of the novel XP molecular recognition and water scoring in separating active and inactive ligands and avoiding false positives.

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Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes

Networks of interacting biomolecules carry out many essential functions in living cells, but the 'design principles' underlying the functioning of such intracellular networks remain poorly understood, despite intensive efforts including quantitative analysis of relatively simple systems Here we present a complementary approach to this problem: the design and construction of a synthetic network to implement a particular function We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, termed the repressilator, in Escherichia coli The network periodically induces the synthesis of green fluorescent protein as a readout of its state in individual cells The resulting oscillations, with typical periods of hours, are slower than the cell-division cycle, so the state of the oscillator has to be transmitted from generation to generation This artificial clock displays noisy behaviour, possibly because of stochastic fluctuations of its components Such 'rational network design may lead both to the engineering of new cellular behaviours and to an improved understanding of naturally occurring networks

/pdf/a-synthetic-oscillatory-network-of-transcriptional-1c0xh9y2s2.pdf

A synthetic oscillatory network of transcriptional regulators

In the AAA+ ClpXP protease, ClpX uses repeated cycles of ATP hydrolysis to pull native proteins apart and to translocate the denatured polypeptide into ClpP for degradation. Here, we probe polypeptide features important for translocation. ClpXP degrades diverse synthetic peptide substrates despite major differences in side-chain chirality, size, and polarity. Moreover, translocation occurs without a peptide -NH and with 10 methylenes between successive peptide bonds. Pulling on homopolymeric tracts of glycine, proline, and lysine also allows efficient ClpXP degradation of a stably folded protein. Thus, minimal chemical features of a polypeptide chain are sufficient for translocation and protein unfolding by the ClpX machine. These results suggest that the translocation pore of ClpX is highly elastic, allowing interactions with a wide range of chemical groups, a feature likely to be shared by many AAA+ unfoldases.

Polypeptide translocation by the AAA+ ClpXP protease machine

Many mutant variants of the P22 Arc repressor are subject to intracellular proteolysis in Escherichia coli, which precludes their expression at levels sufficient for purification and subsequent biochemical characterization. Here we examine the effects of several different C-terminal extension sequences on the expression and activity of a set of Arc mutants. We show that two tail sequences, KNQHE (st5) and H6KNQHE (st11), increase the expression levels of most mutants from 10- to 20-fold and, in some cases, result in restoration of biological activity in the cell. A third tail sequence, HHHHHH (st6), was not as effective in increasing mutant expression levels. All three tail sequences are functionally and structurally silent, as judged by their lack of effects on the DNA binding activity and stability of otherwise wild-type Arc. The properties of the st11 tail sequence make it an efficient system for the expression and purification of mutant Arc proteins, both because mutant expression levels are increased and because the proteins can be rapidly purified using nickel-chelate affinity chromatography. Arc mutants containing the EA28, RL31, and SA32 mutations were purified in the st11 background. The thermodynamic stability of the EA28 mutant (delta delta Gu approximately -0.4 kcal/mol) is reduced modestly compared to the st11 parent, whereas the RL31 mutant (delta delta Gu approximately -3.0 kcal/mol) and SA32 mutant (delta delta Gu approximately -3.3 kcal/mol) are substantially less stable.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/pro.5560021219

P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

Publisher Summary This chapter discusses the λ repressor and relates this information to the broader problems of macromolecular recognition, the relationship between protein sequence and structure, and prospects for the rational engineering of stability and binding specificity. The biological activities of λ repressor are overviewed. The λ repressor has provided a useful model system for the study of protein–DNA interactions and gene regulation. Studies of repressor have also addressed basic questions about subunit oligomerization, protein folding and stability, and self-processing via proteolytic cleavage. Small amounts of radioactive A repressor were initially isolated from phage-infected cells and shown to bind specifically to operator DNA. Biochemical studies established that repressor was a single polypeptide of about 26 kDa that the dimer was the active DNA-binding species, and that repressor could aggregate to form higher oligomers. It is essential to have more detailed structural information about the N-terminal arm, becausethis plays such a crucial role in recognition of the λ operators. It is important to determine the structure of a complex with nonoperator DNA arid to determine the structure of the C-terminal domain. The chapter also discusses the C-terminal domain, thermodynamic and kinetic aspects of repressor, and recognition of operator DNA.

λ Repressor: A Model System for Understanding Protein–DNA Interactions and Protein Stability

By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Arc repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper−phenanthroline footprinting studies, but the covalent attachment of the two...

Robert T. Sauer

Papers

Polypeptide translocation by the AAA+ ClpXP protease machine

P22 Arc repressor: Enhanced expression of unstable mutants by addition of polar C‐terminal sequences

λ Repressor: A Model System for Understanding Protein–DNA Interactions and Protein Stability

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

Altered specificity of a AAA+ protease.