608 research outputs found
Thirty years of molecular dynamics simulations on posttranslational modifications of proteins
Posttranslational modifications (PTMs) are an integral component to how cells
respond to perturbation. While experimental advances have enabled improved PTM
identification capabilities, the same throughput for characterizing how
structural changes caused by PTMs equate to altered physiological function has
not been maintained. In this Perspective, we cover the history of computational
modeling and molecular dynamics simulations which have characterized the
structural implications of PTMs. We distinguish results from different
molecular dynamics studies based upon the timescales simulated and analysis
approaches used for PTM characterization. Lastly, we offer insights into how
opportunities for modern research efforts on in silico PTM characterization may
proceed given current state-of-the-art computing capabilities and
methodological advancements.Comment: 64 pages, 11 figure
From Mollusks to Medicine: A Venomics Approach for the Discovery and Characterization of Therapeutics from Terebridae Peptide Toxins
Animal venoms comprise a diversity of peptide toxins that manipulate molecular targets such as ion channels and receptors, making venom peptides attractive candidates for the development of therapeutics to benefit human health. However, identifying bioactive venom peptides remains a significant challenge. In this review we describe our particular venomics strategy for the discovery, characterization, and optimization of Terebridae venom peptides, teretoxins. Our strategy reflects the scientific path from mollusks to medicine in an integrative sequential approach with the following steps: (1) delimitation of venomous Terebridae lineages through taxonomic and phylogenetic analyses; (2) identification and classification of putative teretoxins through omics methodologies, including genomics, transcriptomics, and proteomics; (3) chemical and recombinant synthesis of promising peptide toxins; (4) structural characterization through experimental and computational methods; (5) determination of teretoxin bioactivity and molecular function through biological assays and computational modeling; (6) optimization of peptide toxin affinity and selectivity to molecular target; and (7) development of strategies for effective delivery of venom peptide therapeutics. While our research focuses on terebrids, the venomics approach outlined here can be applied to the discovery and characterization of peptide toxins from any venomous taxa
Unnatural Amino Acids Improve Affinity and Modulate Immunogenicity: Developing Peptides to Treat MHC Type II Autoimmune Disorders
Many autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (RA), and celiac disease (CD), arise from improper immune system recognition of self or benign peptides as threats. No autoimmune disease currently has a cure. Many treatments suppress the entire immune system to decrease symptom severity. The core molecular interaction underlying these diseases involves specific alleles of the human leukocyte antigen (HLA) receptor hosting the immunodominant peptides associated with the disease (i.e. myelin basic protein, Type II collagen, or α-gliadin) in their binding groove. Once bound, circulating T-cells can recognize the HLA-antigen complex and initiate the complex cascade that forms an adaptive immune response. This initial HLA-antigen interaction is a promising target for therapeutic intervention. Two general strategies have been pursued: altered peptide ligands (APLs) that attempt to recruit a different class of T-cell to induce an anti-inflammatory response to balance the pro-inflammatory response associated with the antigen; and HLA blockers (HLABs), peptides that, due to a much higher affinity for the HLA receptor, quantitatively displace the antigen, inhibiting the immune response. Both approaches would benefit from improved HLA-drug binding, but as the HLA receptors are highly promiscuous, the binding sites are not specific for any natural amino acid. Unnatural amino acids, either designed or screened through high-throughput assays, may provide a solution. This review summarizes the nascent field of using non-canonical residues to treat MS, RA and CD, focusing on the importance of specific molecular interactions, and provides some examples of the synthesis of these unnatural residues
Structural Basis for the Site-Specific Incorporation of Lysine Derivatives into Proteins
Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated epsilon-N-alkynyl-,epsilon-N-butyryl-,epsilon-N-crotonyl- and epsilon-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance
Extending enzyme molecular recognition with an expanded amino acid alphabet
Natural enzymes are constructed from the twenty proteogenic amino acids, which may then require post-translational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of non-canonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different non-canonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically-modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modelling studies revealed that the unique shape and functionality of the non-canonical side chain enabled the active site to be remodelled to enable more efficient stabilisation of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties
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Engineering the central dogma using emulsion based directed evolution
The central dogma of molecular biology forms the most basic (and fundamental) paradigm of how life operates. Despite its elegant simplicity, scientists are still uncovering enigmas of the central dogma - which has been shaped throughout billions of years of the Darwinian process. Even though the core concepts of the central dogma have largely been untouched by evolution (the universality of the genetic code, amino acid utilization, DNA/RNA base identity) scientific advances have shown that these fundamental properties can be altered dramatically. This implies the architectures of life are pliable and likely the result of extreme optimization and fine tuning of semi-random events that took place soon after the origin of life. Reengineering the parameters of life offers a unique way of testing evolutionary processes and perceived optimality of its components. Naturally, coaxing proteins and nucleic acids to function in an unnatural fashion is difficult. Development of techniques to enable these changes has relied heavily on the exploitation of water-in-oil emulsions (or, in vitro compartmentalization), which allows directed evolution at the single cell or even single molecule level. In particular, compartmentalized partnered replication (CPR) is a dual mode selection technique, coupling the in vivo functionality of a gene with the in vitro amplification via emulsion PCR. The CPR technique has enabled the development of synthetic promoter recognition by T7 RNA polymerase, unnatural amino acid incorporation by aminoacyl tRNA synthetase engineering, genetic code reassignment through tRNA evolution, and transcriptional regulation using repressors with novel allosteric effector molecules and operator binding sites. Using a similar technique, the template recognition of an Archaeal DNA polymerase was altered such that the polymerase utilizes both DNA and RNA templates with similar efficiencies. This resulted in a reverse transcriptase that can functionally proofread on RNA templates. These technologies will continue to play a pivotal role in the future development of particular aspects of the central dogma. As certain steps in this process are tweaked to have alternative functionalities and combined together, the gap between natural life and synthetically modified life widens and gives the Darwinian process of evolution new areas to explore.Cellular and Molecular Biolog
Site-directed labelling of proteins for NMR and EPR studies
Site-specific protein labelling presents an important tool for
protein structural biology by
spectroscopic techniques. This thesis focuses on the development
of new spectroscopic
labels and labelling strategies to improve the sensitivity,
accuracy and scope of NMR and
EPR spectroscopy experiments of proteins.
Double electron–electron resonance (DEER) spectroscopy measures
the distance
between two paramagnetic metal centres introduced by
site-specific attachment of
suitable tag molecules. Good DEER tags possess rigid tethers to
position their
paramagnetic centres at a well-defined location relative to the
protein and deliver narrow
DEER distance distributions with high sensitivity, which can
provide accurate
information about protein flexibility. This thesis introduces
new, cyclen-based Gd 3+ tags
and small, Gd 3+ chelating tags designed to deliver narrow DEER
distance distributions.
Chapter 2 describes the development of two cyclen-based
double-arm Gd 3+ tags
designed for binding to the target protein at two and three
points to obtain the narrowest
Gd 3+ –Gd 3+ DEER distance distributions ever recorded with
proteins. It also describes
DEER distance measurements with the iminodiacetic acid tag
attached to cysteine (Cys),
where tags attached to two neighbouring Cys residues combine to
chelate a single Gd 3+
ion. These results have been published in a journal article
(Welegedara et al., Chem. Eur.
J. 2017, 23, 11694−11702).
Chapter 3 discusses two single-armed Gd 3+ tags, a cyclen-based
Gd 3+ tag and a
PyMTA tag that forms a heptadentate Gd 3+ binding motif. Both
tags deliver the shortest
possible tethers to cysteine residues and are shown to produce
narrow DEER distance
distributions in proteins.
For applications in NMR spectroscopy, proteins can be labelled
site-specifically
with NMR probes such as trimethylsilyl (TMS) probes, which
deliver readily detectable
1D 1 H-NMR signals. Introduction of paramagnetic tags and NMR
probes by attachment
to Cys residues requires mutations of native Cys residues to
achieve site-selectivity,
which is not possible with structurally and functionally
important Cys residues. Chapter
4 demonstrates a solution to this limitation by introducing a
selenocysteine (Sec) residue,
which can be selectively reacted with probe molecules at slightly
acidic pH without
iiiaffecting naturally occuring Cys residues. To achieve this, a
Sec residue was introduced
as a photocaged unnatural amino acid (UAA), PSc, to bypass the
otherwise unavoidable
challenges associated with the natural Sec incorporation
mechanism. UV illumination of
PSc yielded Sec with no evidence for the formation of undesired
dehydroalanine
byproducts. Selective tagging of Sec residues with TMS tags was
shown to deliver a
useful tool for studies of ligand binding to proteins. These
results have also been
published in a journal article (Welegedara et al., Bioconjugate
Chem. 2018, 19,
2257−2264).
Site-selective incorporation of isotope-labelled PSc, photolysis
and anaerobic
deselenization opens an indirect route to labelling a single
specific alanine residue in a
protein with stable isotopes. Such samples would have important
applications in
heteronuclear NMR, as they allow the selective detection of the
labelled alanine residue
with maximal spectral resolution. As shown in Chapter 5,
deselenization of
selenoproteins into alanine is possible but requires extremely
anaerobic conditions to
eliminate serine as the main unwanted byproduct.
A range of UAAs has been developed to serve as spectroscopic
probes or facilitate
the introduction of spectroscopic probes via biorthogonal
reactions. The increasing
demand for proteins with different UAAs and the significant cost
of some of these UAAs
has led to increasing popularity of cell-free protein synthesis
(CFPS) systems, which use
amino acids more sparingly than in vivo expression systems.
Mutants of pyrrolysyl-tRNA
synthetase (PylRS)/tRNA CUA pairs have been developed into a
particularly versatile tool
for the incorporation of many structurally different UAAs, but
most produce
disappointedly poor protein yields in vivo. Chapter 6 describes
attempts to develop an in-
house CFPS system with a PylRS/tRNA CUA pair. In addition, the
polyspecific G2
synthetase has been reported to facilitate the incorporation of
sterically demanding UAAs
and Chapter 7 of this thesis describes attempts to develop a CFPS
system with the G2
synthetase
The Development Of Unnatural Amino Acid-Based Probes And Methods For Biological Studies
Proteins form a diverse ensemble of dynamic structures to carry out all life-sustaining functions. Therefore, many efforts have gone into studying the structure-dynamics-function relationship of proteins using a wide range of techniques, including fluorescence and infrared (IR) spectroscopies. While very useful, intrinsic fluorescence and IR signals arising from the natural amino acid side chains within the protein are often insufficient or unable to provide the information needed to understand the biological question of interest. To this end, various extrinsic spectroscopic probes, such as fluorescent dyes, have been used to increase the information content in specific measurements and applications. However, incorporation of a foreign moiety into any protein unavoidably affects its native structure and dynamics; hence effort must be made to reduce such perturbation. In this regard, the overarching aim of this thesis is to develop novel spectroscopic probes based on scaffolds of natural amino acids (NAAs). Because of their small size and similarity to NAAs, such unnatural amino acid-based (UAA-based) probes are expected to be minimally perturbing. Specifically, we show that (1) 4-cyanotryptophan (4CN-Trp) is a blue fluorescent amino acid useful for fluorescence microscopy applications; (2) 4CN-Trp and DiO (a common dye used to stain membranes) are a useful FRET pair to study peptide-membrane interactions; (3) 4CN-Trp, and tryptophan constitutes a dual FRET-PET pair which was used to study peptide end-to-end termini interactions and protein ligand-binding; and (4) the functional group of 4CN-Trp, 4-cyanoindole can be used in the form of a nucleoside as a dual fluorescence-IR reporter for DNA-protein studies. Furthermore, we extended applications of previously known UAAs and showed (5) p-cyanophenylalanine is useful as a fluorescence-based pH sensor which we used to determine peptide pKa’s and peptide membrane penetration kinetics and (6) we use a simple synthetic method for post-translationally installing an ester moiety on to proteins via cysteine alkylation as an UAA-based vibrational probe in proteins to study fibril formation and protein-ligand interaction
Using a reprogrammed genetic code to modulate protein activity by novel post-translational control
Despite the diverse structures and functions sampled by the proteome, all proteins comprise 20 canonical amino acids that sample only a small percentage of available chemistry. This limitation is lifted somewhat through the use of post-translational modifications, however the limit imposed by the restricted number of amino acids inherently limits the variety of protein function and control that can be accessed. One powerful route to diversify the chemistry sampled by proteins is through genetically encoded unnatural amino acid (uAA) incorporation. The uAA p-azido-L-phenylalanine (AzPhe) can introduce two novel methods of control, photochemical covalent rearrangement and Click chemistry. AzPhe incorporation combined with these two methods of novel post-translational control were used to modulate the function of two distinct proteins; TEM β-lactamase and sfGFP.
This thesis introduces the use of uAAs and the technical modifications required to enable uAA incorporation in vivo. It describes the in silico approach taken to evaluate potential mutations based on the likelihood of them imparting novel changes to protein function. Nine positions in TEM β-lactamase were chosen for uAA incorporation and the effect on activity was then determined using kinetic analyses. AzPhe incorporation alone resulted in a variety of effects on enzyme activity, ranging from small increases to complete loss of activity. Subsequent post-translational modification using UV light resulted in only slight changes in activity. Modification via Click chemistry using dibenzyl cyclo-octyne (DBCO) derivatives resulted in either inhibition or increased catalytic activity, depending on the position of AzPhe incorporation and the type of adduct used.
Click chemistry was then used to modify TEM β-lactamase with other chemical modifications that enable the immobilization of proteins onto two different surfaces. The π-π stacking interaction between a DBCO-pyrene moiety and graphene was exploited to attach TEM β-lactamase to graphene in a defined and controlled manner, placing the active site in close proximity to the electron cloud of the sp2-bonded material. TEM β-lactamase was then modified using two DNA oligonucleotides that define assembly of a DNA origami “tile”. DNA origami can be used to immobilize multiple proteins at several defined positions, enabling the re-creation of enzyme pathways or signalling cascades in vitro.
Finally, AzPhe was incorporated into sfGFP and the effects of its incorporation and subsequent modification on fluorescence were explored. The incorporation of AzPhe resulted in a blue shifted λmax, a change that was reversed upon UV irradiation. X-ray crystallography suggested that a hydrogen-bonding network involving the chromophore and surrounding residues was disrupted upon AzPhe incorporation, but then reformed upon modification of the uAA. Click chemistry had a variable effect on fluorescence depending on the modification used. Modification of AzPhe with a large fluorescent dye had no effect on the sfGFP fluorescence spectrum, but enabled FRET between the two chromophores. Modification with a DBCO-amine had the same effect as UV irradiation.
Overall, this thesis has shown that the use of genetically encoded uAA incorporation coupled with novel post-translational modifications is a powerful approach for modifying protein function, and facilitating defined interfacing with new and useful materials
Three decades of nucleic acid aptamer technologies: Lessons learned, progress and opportunities on aptamer development
Aptamers are short single-stranded nucleic acid sequences capable of binding to target molecules in a way similar to antibodies. Due to various advantages such as prolonged shelf life, low batch to batch variation, low/no immunogenicity, freedom to incorporate chemical modification for enhanced stability and targeting capacity, aptamers quickly found their potential in diverse applications ranging from therapy, drug delivery, diagnosis, and functional genomics to bio-sensing. Aptamers are generated by a process called SELEX. However, the current overall success rate of SELEX is far from being satisfactory, and still presents a major obstacle for aptamer-based research and application. The need for an efficient selection strategy consisting of defined procedures to deal with a wide variety of targets is significantly important. In this work, by analyzing key aspects of SELEX including initial library design, target preparation, PCR optimization, and single strand DNA separation, we provide a comprehensive analysis of individual steps to facilitate researchers intending to develop personalized protocols to address many of the obstacles in SELEX. In addition, this review provides suggestions and opinions for future aptamer development procedures to address the concerns on key SELEX steps, and post-SELEX modifications
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