Mechanical Unfolding of an Ankyrin Repeat Protein

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly α-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins.     

Expression and Purification of the Crystalline Surface Layer Protein of Rickettsia typhi

The crystalline surface layer (S-layer) protein (SLP) of Rickettsia typhi is known as the protective antigen against murine typhus. We previously reported a cloning and sequence analysis of the SLP gene of R. typhi (slpT) and showed that the open reading frame of this gene encodes both the SLP and a 32-kDa protein. To express only the SLP from this gene, the putative signal sequence and the 32-kDa protein portion were removed from the slpT. This protein was expressed in Escherichia coli as a fusion protein, consisting of the SLP and maltose binding protein. The recombinant protein reacted strongly with polyclonal antiserum of a patient with murine typhus.     

Genetic typing of isolates of Rickettsia typhi

Murine typhus, which is caused by Rickettsia typhi, has a wide range of clinical manifestations. It has a low mortality rate but may result in meningoencephalitis and interstitial pneumonia in severe cases. Comparisons of complete genome sequences of R. typhi isolates from North Carolina, USA (Wilmington), Myanmar (B9991PP), and Thailand (TH1527) identified only 26 single nucleotide polymorphism (SNP) and 7 insertion-deletion (INDEL) sites in these highly syntenic genomes. Assays were developed to further define the distribution of these variant sites among 15 additional isolates of R. typhi with different histories from Asia, the USA, and Africa. Mismatch amplification mutation assays (MAMA) were validated for 22 SNP sites, while the 7 INDEL sites were analyzed directly on agarose gels. Six SNP types, 9 INDEL types, 11 total types were identified among these 18 isolates. Replicate DNA samples as well as comparisons of isolates with different passage and source histories gave consistent genetic typing profiles. Comparison of the SNP and INDEL markers to R. typhi’s nearest neighbor Rickettsia prowazekii demonstrated that the majority of the SNPs represent intra-species variation that arose post divergence of these two species while several INDEL sites also exhibited intraspecies variability among the R. prowazekii genomes that have been completely sequenced. The assays for the presence of these SNP and INDEL sites, particularly the latter, comprise a low technology gel method for consistently distinguishing R. typhi and R. prowazekii as well as for differentiating genetic types of R. typhi.     

Serosurvey of Rickettsia typhi and Rickettsia felis in HIV‐infected patients

Consistent with the effects of HIV on cell‐mediated immunity, an increased susceptibility to intracellular microorganisms has been observed. Rickettsiae are obligate intracellular microorganisms. The aim of this study was to examine Rickettsia typhi and Rickettsia felis infections in HIV+ population. Sera of 341 HIV+ patients were evaluated by indirect immunofluorescent assay. Age, sex, residential locality, risk behavior, stage according to criteria of the Center for Disease Control and Prevention, CD4+/CD8+ T cells, Hepatitis B antigen, and Hepatitis C serology were surveyed. Seroprevalences of R. typhi and R. felis infection were 7.6% and 4.4%, respectively. No associations were found between seropositivities and the assessed variables. Findings were similar to those obtained in healthy subjects from the same region.     

Cross-Reactivity of Rickettsia japonica and Rickettsia typhi Demonstrated by Immunofluorescence and Western Immunoblotting

Cross-reactivity between Rickettsia japonica and R. typhi was observed by immunofluorescence tests using sera from patients with Oriental spotted fever (OSF), from whom the causative agent was isolated and identified as R. japonica. Western immunoblotting with these sera revealed that only the 120-kilodalton surface polypeptide, i.e., rickettsial outer membrane protein (rOmp) B, has a common antigenicity with the 105-kilodalton surface polypeptide of R. typhi. In some cases, antibodies specifically reactive with R. typhi were detected in acute-phase sera followed by a significant rise in titers, possibly because of an anamnestic response to a previous infection with an R. typhi-like agent; the sera retained reactivity to R. typhi even after absorption by a homologous strain. A lipopolysaccharide (LPS)-like antigen of R. typhi was found to be reactive with some sera of OSF patients. The ladder bands on Western immunoblot of rickettsial organisms were confirmed to be polysaccharide in nature, which was demonstrated by comparing them with the pattern of silver-stained gel of proteinase K-treated rickettsial specimens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

植物锚蛋白研究进展

锚蛋白重复序列模体是生物体内最普遍的蛋白质序列模体之一,在多种细胞活动中主要介导蛋白质-蛋白质的相互作用。综述了近年来有关锚蛋白参与植物信号传导的研究进展。     

The Secretion of an Intrinsically Disordered Protein with Different Secretion Signals in Bacillus subtilis

In this study, a naturally unsecretory intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was attempted to be secreted with different types of secretion signals in Bacillus subtilis. The results showed that Nsp can be secreted efficiently by all selected Sec-type signal peptides. Nsp was successfully exported when fused to Tat-type signal peptides but less efficient than Sec-type. The fusion protein with the non-classical extracellular proteins can be detected in the cell and extracellular milieu. This study further demonstrated that the mature protein plays an important role in protein secretion. Moreover, these results indicated that Nsp could be a useful tool to understand the individual roles of mature proteins and signal peptide in protein secretion, to evaluate the effect of conformation of mature proteins on their export pathway when coupled with Tat-type signal peptide, and to seek the signal of non-classical secretory proteins.     

Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis

Abstract Pulsed field gel electrophoresis (PFGE) of Sma I, Mlu I and Sal I digested DNA was used to estimate genome size and perform restriction fragment length polymorphism analysis for Rickettsia prowazekii and Rickettsia typhi . We concluded that the genome of R. prowazekii and R. typhi consisted of a single chromosomal DNA. The total length of DNA of R. prowazekii was 1,106±54 kb and of R. typhi was 1,133±44kb. It was possibleto differentiate two strains of R. prowazekii , Breinl and EVir, by PFGE analysis after Sal I digestion. Restriction fragment length polymorphism analysis did not reveal intraspecies differences between three human isolates and one Xenopsilla cheopis isolate of R. typhi .     

Structural and Energetic Characterization of the Ankyrin Repeat Protein Family

Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily amenable for design, with promising biotechnological and biomedical applications. Studying repeat protein families presents technical challenges due to the high sequence divergence among the repeating units. We developed and applied a systematic method to consistently identify and annotate the structural repetitions over the members of the complete Ankyrin Repeat Protein Family, with increased sensitivity over previous studies. We statistically characterized the number of repeats, the folding of the repeat-arrays, their structural variations, insertions and deletions. An energetic analysis of the local frustration patterns reveal the basic features underlying fold stability and its relation to the functional binding regions. We found a strong linear correlation between the conservation of the energetic features in the repeat arrays and their sequence variations, and discuss new insights into the organization and function of these ubiquitous proteins.     

Arabidopsis Acyl-CoA-Binding Protein ACBP2 Interacts With an Ethylene-Responsive Element-Binding Protein,AtEBP, via its Ankyrin Repeats

Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.     

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.     

Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae

Rickettsia typhi, the causative agent of murine typhus, is an obligate intracellular bacterium with a life cycle involving both vertebrate and invertebrate hosts. Here we present the complete genome sequence of R. typhi (1,111,496 bp) and compare it to the two published rickettsial genome sequences: R. prowazekii and R. conorii. We identified 877 genes in R. typhi encoding 3 rRNAs, 33 tRNAs, 3 noncoding RNAs, and 838 proteins, 3 of which are frameshifts. In addition, we discovered more than 40 pseudogenes, including the entire cytochrome c oxidase system. The three rickettsial genomes share 775 genes: 23 are found only in R. prowazekii and R. typhi, 15 are found only in R. conorii and R. typhi, and 24 are unique to R. typhi. Although most of the genes are colinear, there is a 35-kb inversion in gene order, which is close to the replication terminus, in R. typhi, compared to R. prowazekii and R. conorii. In addition, we found a 124-kb R. typhi-specific inversion, starting 19 kb from the origin of replication, compared to R. prowazekii and R. conorii. Inversions in this region are also seen in the unpublished genome sequences of R. sibirica and R. rickettsii, indicating that this region is a hot spot for rearrangements. Genome comparisons also revealed a 12-kb insertion in the R. prowazekii genome, relative to R. typhi and R. conorii, which appears to have occurred after the typhus (R. prowazekii and R. typhi) and spotted fever (R. conorii) groups diverged. The three-way comparison allowed further in silico analysis of the SpoT split genes, leading us to propose that the stringent response system is still functional in these rickettsiae.