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

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A synthetic oscillatory network of transcriptional regulators

In a lysogen, most genes of phage lambda are repressed; in response to a transient induction signal, they are efficiently switched on. The switch, which consists in part of a tripartite operator to which two regulatory proteins bind, depends not only on DNA--protein interactions, but also on effects transmitted from one DNA-bound protein to another. lambda Exemplifies a strategy that facilitates efficient switching between two physiological states in response to a transient signal.

lambda Repressor and cro--components of an efficient molecular switch.

We have generated a series of variable-strength, constitutive, bacterial promoters that act predictably in different sequence contexts, span two orders of magnitude in strength and contain convenient sites for cloning and the introduction of downstream open-reading frames. Importantly, their design insulates these promoters from the stimulatory or repressive effects of many 5 0 -o r 3 0 -sequence elements. We show that different promoters from our library produce constant relative levels of two different proteins in multiple genetic contexts. This set of promoters should be a useful resource for the synthetic-biology community.

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Design, construction and characterization of a set of insulated bacterial promoters

Autoregulation and function of a repressor in bacteriophage lambda.

Events that stall bacterial protein synthesis activate the ssrA-tagging machinery, resulting in resumption of translation and addition of an 11-residue peptide to the carboxyl terminus of the nascent chain. This ssrA-encoded peptide tag marks the incomplete protein for degradation by the energy-dependent ClpXP protease. Here, a ribosome-associated protein, SspB, was found to bind specifically to ssrA-tagged proteins and to enhance recognition of these proteins by ClpXP. Cells with an sspB mutation are defective in degrading ssrA-tagged proteins, demonstrating that SspB is a specificity-enhancing factor for ClpXP that controls substrate choice.

https://science.sciencemag.org/content/sci/289/5488/2354.full.pdf

A specificity-enhancing factor for the ClpXP degradation machine.

Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 μM–10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined. In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.

https://www.pnas.org/content/pnas/95/11/5929.full.pdf

Robert T. Sauer

Papers

lambda Repressor and cro--components of an efficient molecular switch.

Design, construction and characterization of a set of insulated bacterial promoters

Autoregulation and function of a repressor in bacteriophage lambda.

A specificity-enhancing factor for the ClpXP degradation machine.

Optimizing the stability of single-chain proteins by linker length and composition mutagenesis