residue-specific-exchange-proline-proline-analogs-fluorescent

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

University of Manitoba

Can we make life with a different genetic amino acid repertoire? Can we expect organisms which would keep newly given genetic code associations permanently? To address these questions, we would like to analyze the existent genetic code amino acid repertoire as formed from derivatives of alanine. Derivation from alanine leads to the α-helix based biological world, the Alanine World, whereas variations in the side-chains enable tertiary folding and subsequent chemical versatility of the proteome. Proline, glycine and pyrrolysine are the rudiments in the current genetic code, indicating that the original set could be different. Furthermore, from the perspective of peptide chemistry, it shall be possible to recruit these alternative scaffolds for the construction of synthetic or alternative life. This would allow for a completely new biological world, potentially as functional and versatile as the existing one. Pursuing these options offers a strategy for a complete re-design or even de-novo creation of living organisms based on entirely different chemical make-up, with completely new set of solutions for both near and distant future biotechnologies.

Anticipating alien cells with alternative genetic codes: away from the alanine world!

Collagen mimics are peptides designed to reproduce structural features of natural collagen. A triple helix is the first element in the hierarchy of collagen folding. It is an assembly of three parallel peptide chains stabilized by packing and interchain hydrogen bonds. In this review we summarize the existing chemical approaches towards stabilization of this structure including the most recent developments. Currently proposed methods include manipulation of the amino acid composition, application of unnatural amino acid analogues, stimuli-responsive modifications, chain tethering approaches, peptide amphiphiles, modifications that target interchain interactions and more. This ability to manipulate the triple helix as a supramolecular self-assembly contributes to our understanding of the collagen folding. It also provides essential information needed to design collagen-based biomaterials of the future.

Stabilization of the triple helix in collagen mimicking peptides.

Our understanding of protein folds relies fundamentally on the set of secondary structures found in the proteomes. Yet, there also exist intriguing structures and motifs that are underrepresented in natural biopolymeric systems. One example is the polyproline II helix, which is usually considered to have a polar character and therefore does not form membrane spanning sections of membrane proteins. In our work, we have introduced specially designed polyproline II helices into the hydrophobic membrane milieu and used 19F NMR to monitor the helix alignment in oriented lipid bilayers. Our results show that these artificial hydrophobic peptides can adopt several different alignment states. If the helix is shorter than the thickness of the hydrophobic core of the membrane, it is submerged into the bilayer with its long axis parallel to the membrane plane. The polyproline helix adopts a transmembrane alignment when its length exceeds the bilayer thickness. If the peptide length roughly matches the lipid thickness, a coexistence of both states is observed. We thus show that the lipid thickness plays a determining role in the occurrence of a transmembrane polyproline II helix. We also found that the adaptation of polyproline II helices to hydrophobic mismatch is in some notable aspects different from α-helices. Finally, our results prove that the polyproline II helix is a competent structure for the construction of transmembrane peptide segments, despite the fact that no such motif has ever been reported in natural systems.

Bilayer thickness determines the alignment of model polyproline helices in lipid membranes.

A central question in the evolution of the modern translation machinery is the origin and chemical ethology of the amino acids prescribed by the genetic code. The RNA World hypothesis postulates that templated protein synthesis has emerged in the transition from RNA to the Protein World. The sequence of these events and principles behind the acquisition of amino acids to this process remain elusive. Here we describe a model for this process by following the scheme previously proposed by Hartman and Smith, which suggests gradual expansion of the coding space as GC-GCA-GCAU genetic code. We point out a correlation of this scheme with the hierarchy of the protein folding. The model follows the sequence of steps in the process of the amino acid recruitment and fits well with the co-evolution and coenzyme handle theories. While the starting set (GC-phase) was responsible for the nucleotide biosynthesis processes, in the second phase alanine-based amino acids (GCA-phase) were recruited from the core metabolism, thereby providing a standard secondary structure, the α-helix. In the final phase (GCAU-phase), the amino acids were appended to the already existing architecture, enabling tertiary fold and membrane interactions. The whole scheme indicates strongly that the choice for the alanine core was done at the GCA-phase, while glycine and proline remained rudiments from the GC-phase. We suggest that the Protein World should rather be considered the Alanine World, as it predominantly relies on the alanine as the core chemical scaffold.

The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis.

Xenobiology is the science of estranged life forms. More specifically, this is an emergent technoscience that combines advances in genetic engineering with the design of biological systems based on unusual biochemistries delivered by chemical compounds of mostly anthropogenic origin. Xenobiology enables us to create and study strange new life forms, "aliens", not in the way science fiction books do it, but in terms of enlightened science, design, and engineering.

Xenobiology: A Journey towards Parallel Life Forms.

Fluorine-containing analogues of proline are valuable tools in engineering and NMR spectroscopic studies of peptides and proteins. Their use relies on the fundamental understanding of the interplay between the substituents and the main chain groups of the amino acid residue. This study aims to showcase the polarity-related effects that arise from the interaction between the functional groups in molecular models. Properties such as conformation, acid-base transition, and amide-bond isomerism were examined for diastereomeric 4-fluoroprolines, 4-(trifluoromethyl)prolines, and 1,1-difluoro-5-azaspiro[2.4]heptane-6-carboxylates. The preferred conformation on the proline ring originated from a preferential axial positioning for a single fluorine atom, and an equatorial positioning for a trifluoromethyl- or a difluoromethylene group. This orientation of the substituents explains the observed trends in the p  values, lipophilicity, and the kinetics of the amide bond rotation. The study also provides a set of evidences that the transition state of the amide-bond rotation in peptidyl-prolyl favors C- conformation of the pyrrolidine ring.

Polarity effects in 4-fluoro- and 4-(trifluoromethyl)prolines.

Genetic code engineering aims to produce organisms that translate genetic information in a different way from that prescribed by the standard genetic code. This endeavor could eventually lead to genetic isolation, where an organism that operates under a different genetic code will not be able to transfer functional genes with other living species, thereby standing behind a genetic firewall. It is not clear however, how distinct the code should be, or how to measure the distance. We have developed a metric (Δ ) where we assigned polarity indices (clog D ) to amino acids to calculate the distances between pairs of genetic codes. We then calculated the distance between a set of 204 genetic codes, including the 24 known distinct natural codes, 11 extreme-distance codes created computationally, nine theoretical special purpose codes from literature and 160 codes in which canonical amino acids were replaced by noncanonical chemical analogues. The metric can be used for building strategies towards creating semantically alienated organisms, and testing the strength of genetic firewalls. This metric provides the basis for a map of the genetic codes that could guide future efforts towards novel biochemical worlds, biosafety and deep barcoding applications.

How To Quantify a Genetic Firewall? A Polarity-Based Metric for Genetic Code Engineering.

Coupling functional moieties to lantibiotics offers exciting opportunities to produce novel derivatives with desirable properties enabling new functions and applications. Here, five different synthetic hydrophobic polyproline peptides were conjugated to either nisin AB (the first two rings of nisin) or nisin ABC (the first three rings of nisin) by using click chemistry. The antimicrobial activity of nisin ABC + O6K3 against  decreased 8-fold compared to full-length nisin, but its activity was 16-fold better than nisin ABC, suggesting that modifying nisin ABC is a promising strategy to generate semi-synthetic nisin hybrids. In addition, the resulting nisin hybrids are not prone to degradation at the C-terminus, which has been observed for nisin as it can be degraded by nisinase or other proteolytic enzymes. This methodology allows for getting more insight into the possibility of creating semi-synthetic nisin hybrids that maintain antimicrobial activity, in particular when synthetic and non-proteinaceous moieties are used. The success of this approach in creating viable nisin hybrids encourages further exploring the use of different modules, e.g., glycans, lipids, active peptide moieties, and other antimicrobial moieties.

Conjugation of Synthetic Polyproline Moietes to Lipid II Binding Fragments of Nisin Yields Active and Stable Antimicrobials.

Organofluorine compounds are known to be toxic to a broad variety of living beings in different habitats, and chemical fluorination has been historically exploited by mankind for the development of therapeutic drugs or agricultural pesticides. On the other hand, several studies so far have demonstrated that, under appropriate conditions, living systems (in particular bacteria) can tolerate the presence of fluorinated molecules (e.g., amino acids analogues) within their metabolism and even repurpose them as alternative building blocks for the synthesis of cellular macromolecules such as proteins. Understanding the molecular mechanism behind these phenomena would greatly advance approaches to the biotechnological synthesis of recombinant proteins and peptide drugs. However, information about the metabolic effects of long-term exposure of living cells to fluorinated amino acids remains scarce. Hereby, we report the long-term propagation of  () in an artificially fluorinated habitat that yielded two strains naturally adapted to live on fluorinated amino acids. In particular, we applied selective pressure to force a tryptophan (Trp)-auxotrophic strain to use either 4- or 5-fluoroindole as essential precursors for the  synthesis of Trp analogues, followed by their incorporation in the cellular proteome. We found that full adaptation to both fluorinated Trp analogues requires a low number of genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity, and quality control of protein folding. These findings highlight the cellular mechanisms behind the adaptation to unnatural amino acids and provide the molecular foundation for bioengineering of novel microbial strains for synthetic biology and biotechnology.

Multiomics Analysis Provides Insight into the Laboratory Evolution of  toward the Metabolic Usage of Fluorinated Indoles.

Due to the heterocyclic structure and distinct conformational profile, proline is unique in the repertoire of the 20 amino acids coded into proteins. Here, we summarize the biochemical work on the replacement of proline with (4)- and (4)-fluoroproline as well as 4,4-difluoroproline in proteins done mainly in the last two decades. We first recapitulate the complex position and biochemical fate of proline in the biochemistry of a cell, discuss the physicochemical properties of fluoroprolines, and overview the attempts to use these amino acids as proline replacements in studies of protein production and folding. Fluorinated proline replacements are able to elevate the protein expression speed and yields and improve the thermodynamic and kinetic folding profiles of individual proteins. In this context, fluoroprolines can be viewed as useful tools in the biotechnological toolbox. As a prospect, we envision that proteome-wide proline-to-fluoroproline substitutions could be possible. We suggest a hypothetical scenario for the use of laboratory evolutionary methods with fluoroprolines as a suitable vehicle to introduce fluorine into living cells. This approach may enable creation of synthetic cells endowed with artificial biodiversity, containing fluorine as a bioelement.

Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement.

Among other features, the polarity of amino acid residues is the key parameter for understanding their role in proteins. The wide occurrence of protein modifications in nature and the advent of genetic code engineering techniques created a need for an experimental polarity value integrating both coded (canonical) and noncoded (noncanonical) residues on one universal scale. To address this issue, this work reports on a polarity scale based on the experimental lipophilicity of methyl esters of N-acetylamino acids. The derivatization of amino acids was performed in two steps under mild conditions that allowed conversion of a wide array of amino acids into analytical derivatives. The partitioning/distribution between octan-1-ol and water/buffer was measured using the intensity of the NMR signal as a characteristic for the concentration. The reference set of twenty coded amino acids generated log P values spanning 5.1 units: from tryptophan being the most hydrophobic to aspartate being the most hydrophilic. Furthermore, lipophilicity was measured for a set of analogues of phenylalanine, tyrosine, tryptophan, methionine, proline, and lysine that are typical in nature and/or laboratory practice. The polarity scale reported here will aid the rationalization of amino acid replacements in proteins, and will guide further efforts in experimental genetic code engineering.

Experimental lipophilicity scale for coded and noncoded amino acid residues.

Circular dichroism is a conventional method for studying the secondary structures of peptides and proteins and their transitions. While certain circular dichroism features are characteristic of α-helices and β-strands, the third most abundant secondary structure, the polyproline-II helix, does not exhibit a strictly conserved spectroscopic appearance. Due to its extended nature, the polyproline-II helix is highly accessible to the surrounding solvent; thus, the environment has a critical influence on the lineshape of the circular dichroism spectra of this structure. To showcase possible effects due to the medium, in this work, we report an experimental spectroscopic study of polyproline-II-forming oligomeric peptides in various environments: solvents, detergent micelles, and liposomes. Strikingly, the examination of an oligomeric peptide in a solvent series showed a remarkable 7 nm solvatochromic shift in the main negative band starting with hexafluoropropan-2-ol and moving to hexane. Furthermore, a previously predicted positive band below 200 nm was discovered in the spectra in nonpolar environments. In isotropic liposomes, the expected transition to the transmembrane state correlated with the appearance of a positive band at 228 nm. Our results demonstrate that changes in solvation should be taken into consideration when assessing the circular dichroism spectra of peptides expected to adopt the polyproline-II conformation. Although this precaution may complicate spectral analysis, characterization of solvent-induced spectral changes can generate new opportunities for testing the location of peptides in complex systems such as micelles or lipid bilayers.

Article	Year
Anticipating alien cells with alternative genetic codes: away from the alanine world! Current opinion in biotechnology\| PubMed ID: 31279217	2019
Stabilization of the triple helix in collagen mimicking peptides. Organic & biomolecular chemistry\| PubMed ID: 31464337	2019
Bilayer thickness determines the alignment of model polyproline helices in lipid membranes. Physical chemistry chemical physics : PCCP\| PubMed ID: 31577299	2019
The Alanine World Model for the Development of the Amino Acid Repertoire in Protein Biosynthesis. International journal of molecular sciences\| PubMed ID: 31694194	2019
Xenobiology: A Journey towards Parallel Life Forms. Chembiochem : a European journal of chemical biology\| PubMed ID: 32323410	2020
Polarity effects in 4-fluoro- and 4-(trifluoromethyl)prolines. Beilstein journal of organic chemistry\| PubMed ID: 32765799	2020
How To Quantify a Genetic Firewall? A Polarity-Based Metric for Genetic Code Engineering. Chembiochem : a European journal of chemical biology\| PubMed ID: 33231343	2021
Conjugation of Synthetic Polyproline Moietes to Lipid II Binding Fragments of Nisin Yields Active and Stable Antimicrobials. Frontiers in microbiology\| PubMed ID: 33329435	2020
Multiomics Analysis Provides Insight into the Laboratory Evolution of toward the Metabolic Usage of Fluorinated Indoles. ACS central science\| PubMed ID: 33532571	2021
Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement. Beilstein journal of organic chemistry\| PubMed ID: 33727970	2021
Experimental lipophilicity scale for coded and noncoded amino acid residues. Organic & biomolecular chemistry\| PubMed ID: 34333582	2021
Remarkably high solvatochromism in the circular dichroism spectra of the polyproline-II conformation: limitations or new opportunities? Physical chemistry chemical physics : PCCP\| PubMed ID: 34825904	2021

Vladimir Kubyshkin

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