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The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This method can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

Huntington’s disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (i) multivesicular bodies (MVBs), or amphisomes and (ii) autolysosomes or residual bodies. The organelles were often found in close proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

Huntington’s disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis., Acta Neuropathologica Communications, 9 (1), ISSN:2051-5960

Huntington’s disease is caused by expansion of a polyglutamine (polyQ) domain within exon 1 of the huntingtin gene (Httex1). A popular hypothesis is that the Httex1 protein undergoes sharp conformational changes as the polyQ length exceeds a threshold of 36 residues. We test this hypothesis by combining novel semi-synthesis strategies with state-of-the-art single molecule Förster resonance energy transfer measurements on biologically relevant Httex1 proteins of five different polyQ lengths. Our results, integrated with atomistic simulations, negate the hypothesis of a sharp, polyQ length-dependent change in the structure of monomeric Httex1. Instead, they support a continuous global compaction with increasing polyQ length and this derives from increased prominence of the globular polyQ domain. More specifically, we show that that monomeric Httex1 adopts tadpole-like architectures for polyQ lengths above and beyond the pathological threshold. Additionally, our results suggest that higher order homo- and / or heterotypic interactions within distinct sub-populations of neurons are likely to be the main source of sharp polyQ length-dependencies of HD. These findings pave the way for uncovering the true structural basis of Httex1-mediated neurotoxicity.

The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1–7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties.

Dynamic detection of protein conformational changes at physiological conditions on a minute amount of samples is immensely important for understanding the structural determinants of protein function in health and disease and to develop assays and diagnostics for protein misfolding and protein aggregation diseases. Herein, we experimentally demonstrate the capabilities of a mid-infrared plasmonic biosensor for real-time and in situ protein secondary structure analysis in aqueous environment at nanoscale. We present label-free ultrasensitive dynamic monitoring ofβ-sheet to disordered conformational transitions in a monolayer of the disease-relatedα-synuclein protein under varying stimulus conditions. Our experiments show that the extracted secondary structure signals from plasmonically enhanced amide I signatures in the protein monolayer can be reliably and reproducibly acquired with second derivative analysis for dynamic monitoring. Furthermore, by using a polymer layer we show that our nanoplasmonic approach of extracting the frequency components of vibrational signatures matches with the results attained from gold-standard infrared transmission measurements. By facilitating conformational analysis on small quantities of immobilized proteins in response to external stimuli such as drugs, our plasmonic biosensor could be used to introduce platforms for screening small molecule modulators of protein misfolding and aggregation.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporat...

The synthesis of chemically-modified proteins is a valuable tool for the study of their function.[1] One of the most enabling protein synthesis technologies is native chemical ligation (NCL), in which a peptide thioester is reacted with a peptide bearing an N-terminal Cys to form a native amide bond.[2] In many cases, one of these two fragments can be expressed in E. coli and then ligated to a smaller synthetic peptide so that a large semi-synthetic protein can be made with a minimum of peptide synthesis. This sort of expressed protein ligation (EPL) can be implemented in two ways.[3] For a synthetic C-terminus, the N-terminal region can be expressed as a fusion to an intein and then converted to a thioester for ligation. Likewise, a protein can be expressed with an N-terminal Cys and ligated to a synthetic thioester. EPL is an extremely efficient way of making large quantities of synthetic proteins. However, there are limitations to the method, most notably, the need for Cys at the site of ligation.

Engineered plasmonic nanoantennas with well-defined resonances in Mid-Infrared (Mid-IR) range enable unique possibilities for surface enhanced infrared absorption spectroscopy of biomolecular monolayers, including proteins, for detection, identification and analysis purposes through their distinct vibrational fingerprints. Proteins amide signatures contain vital information on their secondary structural properties which is important in understanding many incurable diseases such as type II diabetes and neurodegenerative disorders, including Alzheimer’s disease. Therefore introducing non-destructive label-free biosensors that enable structural analysis of proteins at their native states and in aqueous solution will provide unique advantageous in applications related to fundamental biology and diseases. In this work, we showed for the first time that plasmonic Mid-IR nanoantennas can sensitively differentiate the secondary structural compositions of protein monolayers both in dry and aqueous environments. Our experimental results with disease-related proteins using Mid-IR plasmonic nanorod antenna arrays demonstrate our platform as a powerful spectroscopic biosensor by detecting conformational variations in random structured monomeric monolayer and cross β-sheet structured fibrillated α-synuclein (α-Syn) protein as well as dominantly native β-sheet streptavidin protein.[1]

We have shown that thioamides can be incorporated into proteins through semi-synthesis and used as probes to monitor structural changes. To date, our methods have required the presence of a cysteine at the peptide ligation site, which may not be present in the native peptide sequence. Here, we present a strategy for the semi-synthesis of thioproteins using homocysteine as a ligation point with subsequent masking as methionine, making the ligation ‘traceless’.

Methods for synthetically manipulating protein structure enable greater flexibility in the study of protein function. Previous characterization of the Escherichia coli aminoacyl tRNA transferase (AaT) has shown that it can modify the N-terminus of a protein with an amino acid from a tRNA or a synthetic oligonucleotide donor. Here, we demonstrate that AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps from readily available starting materials. We have characterized the enzymatic activity of AaT with aminoacyl adenosyl donors and found that reaction products do not inhibit AaT. The use of adenosyl donors removes the substrate limitations imposed by the use of synthetases for tRNA charging and avoids the complex synthesis of an oligonucleotide donor. Thus, our AaT donors increase the potential substrate scope and reaction scale for N-terminal protein modification under conditions that maintain folding.

Labelling of proteins with pairs of fluorophores enables their conformations to be studied; however, complete incorporation of labels in multiple, pre-defined locations is very difficult. Now, a combination of double unnatural amino acid mutagenesis and selective chemical modification offers a general method to achieve this.

Characterization of the amyloidogenic Parkinson's disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This method can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a "click" reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a "clickable magic bullet" strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.

Plasmonic nanoantennas offer new applications in mid-infrared (mid-IR) absorption spectroscopy with ultrasensitive detection of structural signatures of biomolecules, such as proteins, due to their strong resonant near-fields. The amide I fingerprint of a protein contains conformational information that is greatly important for understanding its function in health and disease. Here, we introduce a non-invasive, label-free mid-IR nanoantenna-array sensor for secondary structure identification of nanometer-thin protein layers in aqueous solution by resolving the content of plasmonically enhanced amide I signatures. We successfully detect random coil to cross β-sheet conformational changes associated with α-synuclein protein aggregation, a detrimental process in many neurodegenerative disorders. Notably, our experimental results demonstrate high conformational sensitivity by differentiating subtle secondary-structural variations in a native β-sheet protein monolayer from those of cross β-sheets, which are characteristic of pathological aggregates. Our nanoplasmonic biosensor is a highly promising and versatile tool for in vitro structural analysis of thin protein layers.