The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

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Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form. Irradiation liberates the trapped molecule, permitting targeted perturbation of a biological process. Uncaging technology and fluorescence microscopy are 'optically orthogonal': the former allows control, and the latter, observation of cellular function. Used in conjunction with other technologies (for example, patch clamp and/or genetics), the light beam becomes a uniquely powerful tool to stimulate a selected biological target in space or time. Here I describe important examples of widely used caged compounds, their design features and synthesis, as well as practical details of how to use them with living cells.

http://nathan.instras.com/MyDocsDB/doc-794.pdf

Caged compounds: photorelease technology for control of cellular chemistry and physiology

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.

/pdf/chemically-ubiquitylated-histone-h2b-stimulates-hdot1l-31x20mijry.pdf

Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation

Site-specific installation of methyl-lysine analogues into recombinant histones

Bioorthogonale Chemie – oder: in einem Meer aus Funktionalität nach Selektivität fischen

Biophysical techniques such as fluorescence spectroscopy and nuclear magnetic resonance (NMR) spectroscopy provide a window into the inner workings of proteins. These approaches make use of probes that can either be naturally present within the protein or introduced through a labeling procedure. In general, the more control one has over the type, location and number of probes in a protein, then the more information one can extract from a given biophysical analysis. Recently, two related approaches have emerged that allow proteins to be labeled with a broad range of physical probes. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are both intein-based approaches that permit the assembly of a protein from smaller synthetic and/or recombinant pieces. Here we provide some guidelines for the use of EPL and PTS, and highlight how the dovetailing of these new protein chemistry methods with standard biophysical techniques has improved our ability to interrogate protein function, structure and folding.

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Protein ligation: an enabling technology for the biophysical analysis of proteins

During their intraerythrocytic development, malaria parasites export hundreds of proteins to remodel their host cell. Nutrient acquisition, cytoadherence and antigenic variation are among the key virulence functions effected by this erythrocyte takeover. Proteins destined for export are synthesized in the endoplasmic reticulum (ER) and cleaved at a conserved (PEXEL) motif, which allows translocation into the host cell via an ATP-driven translocon called the PTEX complex. We report that plasmepsin V, an ER aspartic protease with distant homology to the mammalian processing enzyme BACE, recognizes the PEXEL motif and cleaves it at the correct site. This enzyme is essential for parasite viability and ER residence is essential for its function. We propose that plasmepsin V is the PEXEL protease and is an attractive enzyme for antimalarial drug development.

/pdf/plasmepsin-v-licenses-plasmodium-proteins-for-export-into-219jfo0sif.pdf

Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte

To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.

/pdf/ptex-component-hsp101-mediates-export-of-diverse-malaria-hkljstnof9.pdf

PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes

One in four proteins in Plasmodium falciparum contains asparagine repeats. We probed the function of one such 28-residue asparagine repeat present in the P. falciparum proteasome lid subunit 6, Rpn6. To aid our efforts, we developed a regulatable, fluorescent affinity (RFA) tag that allows cellular localization, manipulation of cellular levels, and affinity isolation of a chosen protein in P. falciparum. The tag comprises a degradation domain derived from Escherichia coli dihydrofolate reductase together with GFP. The expression of RFA-tagged proteins is regulated by the simple folate analog trimethoprim (TMP). Parasite lines were generated in which full-length Rpn6 and an asparagine repeat-deletion mutant of Rpn6 were fused to the RFA tag. The knockdown of Rpn6 upon removal of TMP revealed that this protein is essential for ubiquitinated protein degradation and for parasite survival, but the asparagine repeat is dispensable for protein expression, stability, and function. The data point to a genomic mechanism for repeat perpetuation rather than a positive cellular role. The RFA tag should facilitate study of the role of essential genes in parasite biology.

https://www.pnas.org/content/pnas/108/11/4411.full.pdf

Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag.

The proteome of the malarial parasite Plasmodium falciparum has an overabundance of aggregation-prone asparagine repeat-containing proteins. Muralidharan et al. show that Plasmodium Hsp110 protein potently prevents aggregation of asparagine-rich proteins, thereby allowing the parasite to survive febrile episodes.

/pdf/plasmodium-falciparum-heat-shock-protein-110-stabilizes-the-14g2tdegj9.pdf

Vasant Muralidharan

Papers

Protein ligation: an enabling technology for the biophysical analysis of proteins

Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte

PTEX component HSP101 mediates export of diverse malaria effectors into host erythrocytes

Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag.

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