Exploring the Biophysical Properties of Domains Within Multi-Domain Proteins

The past few decades, considerable progress has been made in understanding the biophysical properties of proteins using small modular domains such as SH3 domains. However, there is a surprising lack of knowledge regarding how these properties are affected when the domain is placed back within its full-length multi-domain protein. Using a combination of expressed protein ligation (EPL) and in vivo amino acid replacement of tryptophans with tryptophan (Trp) analogues, we have developed an integrated approach that allows the domain-specific incorporation of optical probes into large recombinant proteins. The Src homology 3 (SH3) domain from the c-Crk-l adaptor protein has been labeled with a Trp analogue, 7-azatryptophan (7AW), using E.coli Trp auxotrophs. Biophysical analysis shows that incorporation of 7AW does not significantly perturb the structure or function of the isolated domain. Ligation of 7AW labeled SH3 domain to the c-Crk-l Src homology 2 (SH2) domain, via EPL, generated the multi-domain protein, c-Crk-l, with a domain specific label. Studies on this labeled protein show that the biochemical and thermodynamic properties of the SH3 domain do not change within the context of a larger multi-domain protein. We have also utilized this technique and segmental isotopic labeling to study the Cterminal SH3 (cSH3) domain of the signaling adaptor protein, Crk-ll. Several studies suggest that the cSH3 domain plays an important regulatory role in the protein. However, no structural information is available on this domain and relatively little is known about its binding partners. We have solved the solution NMR structure of the C-terminal SH3 domain and it adopts the standard SH3 fold comprising a fivestranded p-barrel. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. Studies on the cSH3 domain specifically labeled within Crk-ll have provided the strongest evidence yet that the cSH3 domain interacts in an intramolecular fashion to regulate Crk-ll. The techniques developed here should be applicable to many other multi-domain proteins. This sets the stage for a better understanding of the biophysical properties of domains within these complex systems

Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers

One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. We report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of Asn. PfHsp110c gene knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes

Metabolomics profiling reveals new aspects of dolichol biosynthesis in Plasmodium falciparum

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite’s development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite

Proposed role of <i>P. falciparum</i> Hsp110 and other chaperones as capacitors for evolutionary change.

<p>Malaria parasite proteins with asparagine repeat–containing sequences have a greater risk of aggregation. <i>P. falciparum</i> Hsp110c, possibly with the help of other chaperones, negates much of this risk, thereby allowing these loop-like regions to mutate. Over time these mutations can give rise to novel protein domains, allowing the parasite to develop new functionalities such as drug resistance and new pathogenic factors.</p

Tuning protein autoinhibition by domain destabilization.

Activation of many multidomain signaling proteins requires rearrangement of autoinhibitory interdomain interactions that occlude activator binding sites. In one model for activation, the major inactive conformation exists in equilibrium with activated-like conformations that can be stabilized by ligand binding or post-translational modifications. We established the molecular basis for this model for the archetypal signaling adaptor protein Crk-II by measuring the thermodynamics and kinetics of the equilibrium between autoinhibited and activated-like states. We used fluorescence and NMR spectroscopies together with segmental isotopic labeling by means of expressed protein ligation. The results demonstrate that intramolecular domain-domain interactions both stabilize the autoinhibited state and induce the activated-like conformation. A combination of favorable interdomain interactions and unfavorable intradomain structural changes fine-tunes the population of the activated-like conformation and allows facile response to activators. This mechanism suggests a general strategy for optimization of autoinhibitory interactions of multidomain proteins

Field evaluation of malaria malachite green loop-mediated isothermal amplification in health posts in Roraima state, Brazil

Abstract Background Microscopic detection of malaria parasites is the standard method for clinical diagnosis of malaria in Brazil. However, malaria epidemiological surveillance studies specifically aimed at the detection of low-density infection and asymptomatic cases will require more sensitive and field-usable tools. The diagnostic accuracy of the colorimetric malachite green, loop-mediated, isothermal amplification (MG-LAMP) assay was evaluated in remote health posts in Roraima state, Brazil. Methods Study participants were prospectively enrolled from health posts (healthcare-seeking patients) and from nearby villages (healthy participants) in three different study sites. The MG-LAMP assay and microscopy were performed in the health posts. Two independent readers scored the MG-LAMP tests as positive (blue/green) or negative (clear). Sensitivity and specificity of local microscopy and MG-LAMP were calculated using results of PET-PCR as a reference. Results A total of 91 participants were enrolled. There was 100% agreement between the two MG-LAMP readers (Kappa = 1). The overall sensitivity and specificity of MG-LAMP were 90.0% (95% confidence interval (CI) 76.34–97.21%) and 94% (95% CI 83.76–98.77%), respectively. The sensitivity and specificity of local microscopy were 83% (95% CI 67.22–92.66%) and 100% (95% CI 93.02–100.00%), respectively. PET-PCR detected six mixed infections (infection with both Plasmodium falciparum and Plasmodium vivax); two of these were also detected by MG-LAMP and one by microscopy. Microscopy did not detect any Plasmodium infection in the 26 healthy participants; MG-LAMP detected Plasmodium in five of these and PET-PCR assay detected infection in three. Overall, performing the MG-LAMP in this setting did not present any particular challenges. Conclusion MG-LAMP is a sensitive and specific assay that may be useful for the detection of malaria parasites in remote healthcare settings. These findings suggest that it is possible to implement simple molecular tests in facilities with limited resources

The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle

ABSTRACT Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum. IMPORTANCE Half of the world’s population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite’s ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784–1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC