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1.
Chembiochem ; : e202400148, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38629812

RESUMO

Native chemical ligation is a key reaction in the toolbox of chemical methods for the synthesis of native and modified proteins. The catalysis of ligation is commonly performed by using small aryl-thiol molecules added at high concentrations. In this work, we incorporated thiotyrosine, a non-canonical amino acid containing an aryl-thiol moiety, into a designed cyclic protein « sans queue ni tête ¼. Importantly, the protein environment reduced the pKa of the thiol group to 5.8-5.9, which is significantly lower than the previously reported value for thiotyrosine in a short peptide (pKa 6.4). Furthermore, we demonstrated the catalytic activity of this protein both as hydrolase and in native chemical ligation of peptides. These results will be useful for the development of efficient protein catalysts (enzymes) for protein synthesis and modification.

2.
Biomater Sci ; 12(6): 1590-1602, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38323504

RESUMO

The effects of peptide N- and C-termini on aggregation behavior have been scarcely studied. Herein, we examine (105-115) peptide fragments of transthyretin (TTR) containing various functional groups at both termini and study their impact on the morphology and the secondary structure. We synthesized TTR(105-115) peptides functionalized with α-amino (H-), N-acetyl-α-amino (Ac-) or N,N-dimethyl-α-amino (DiMe-) groups at the N-terminus, and with amide (-NH2) or carboxyl (-OH) functions at the C-terminus. We also investigated quasi-racemic mixtures by mixing the L-enantiomers with the D-enantiomer capped by H- and -NH2 groups. We observed that fibril formation is promoted by the sufficient number of hydrogen bonds at peptides' termini. Moreover, the final morphology of the aggregates can be controlled by the functional groups at the N-terminus. Remarkably, all quasi-racemic mixtures resulted in the robust formation of fibrils. Overall, this work illustrates how modifications of peptide termini may help to engineer supramolecular aggregates with a predicted morphology.


Assuntos
Amiloide , Peptídeos , Peptídeos/química , Amiloide/química , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína
3.
J Phys Chem B ; 125(21): 5502-5510, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34008978

RESUMO

Amyloid fibrils are peptide or protein aggregates possessing a cross-ß-sheet structure. They possess intrinsic fluorescence property, which is still not fully understood. Herein, we compare structural and optical properties of fibrils formed from L- and D-enantiomers of the (105-115) fragment of transthyretin (TTR) and from their racemic mixture. Our results show that autofluorescence of fibrils obtained from enantiomers differs from that of fibrils from the racemic mixture. In order to elucidate the origin of observed differences, we analyzed the structure and morphology of fibrils and showed how variations in ß-sheet organization influence optical properties of fibrils. We clarified the contribution of aromatic rings and the amyloid backbone to the final blue-green emission of fibrils. This work demonstrates how enantiomeric composition of amino acids allows us to modulate the self-assembly and final morphology of well-defined fibrillar bionanostructures with optical properties controlled by supramolecular organization.


Assuntos
Amiloide , Peptídeos , Sequência de Aminoácidos , Conformação Proteica em Folha beta , Estereoisomerismo
4.
J Am Chem Soc ; 143(9): 3330-3339, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33635059

RESUMO

The design of catalytic proteins with functional sites capable of specific chemistry is gaining momentum and a number of artificial enzymes have recently been reported, including hydrolases, oxidoreductases, retro-aldolases, and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation that possesses no stringent restrictions to the amino acid composition at the ligation junction. We report here the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied a minimalist approach to rationally design an oxyanion hole within a small cavity that contains an adjacent thiol nucleophile. The N-terminus of the α-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition-elimination reactions at the acyl group. Cysteine acting as a principal catalytic residue was introduced at the second residue position of the α-helix N-terminus in a designed three-α-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide-αthioesters as acyl-donors, we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation, and peptide ligation.


Assuntos
Domínio Catalítico , Peptídeo Sintases/química , Aciltransferases/síntese química , Aciltransferases/química , Sequência de Aminoácidos , Biocatálise , Cisteína/química , Hidrólise , Cinética , Peptídeo Sintases/síntese química , Peptídeos/síntese química , Conformação Proteica em alfa-Hélice , Engenharia de Proteínas , Especificidade por Substrato
5.
Magn Reson (Gott) ; 2(2): 795-813, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-37905223

RESUMO

Proline homopolymer motifs are found in many proteins; their peculiar conformational and dynamic properties are often directly involved in those proteins' functions. However, the dynamics of proline homopolymers is hard to study by NMR due to a lack of amide protons and small chemical shift dispersion. Exploiting the spectroscopic properties of fluorinated prolines opens interesting perspectives to address these issues. Fluorinated prolines are already widely used in protein structure engineering - they introduce conformational and dynamical biases - but their use as 19F NMR reporters of proline conformation has not yet been explored. In this work, we look at model peptides where Cγ-fluorinated prolines with opposite configurations of the chiral Cγ centre have been introduced at two positions in distinct polyproline segments. By looking at the effects of swapping these (4R)-fluoroproline and (4S)-fluoroproline within the polyproline segments, we were able to separate the intrinsic conformational properties of the polyproline sequence from the conformational alterations instilled by fluorination. We assess the fluoroproline 19F relaxation properties, and we exploit the latter in elucidating binding kinetics to the SH3 (Src homology 3) domain.

6.
Chemistry ; 26(44): 9889-9899, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32364648

RESUMO

The nuclear coactivator binding domain (NCBD) of transcriptional co-regulator CREB-binding protein (CBP) is an example of conformationally malleable proteins that can bind to structurally unrelated protein targets and adopt distinct folds in the respective protein complexes. Here, we show that the folding landscape of NCBD contains an alternative pathway that results in protein aggregation and self-assembly into amyloid fibers. The initial steps of such protein misfolding are driven by intermolecular interactions of its N-terminal α-helix bringing multiple NCBD molecules into contact. These oligomers then undergo slow but progressive interconversion into ß-sheet-containing aggregates. To reveal the concealed aggregation potential of NCBD we used a chemically synthesized mirror-image d-NCBD form. The addition of d-NCBD promoted self-assembly into amyloid precipitates presumably due to formation of thermodynamically more stable racemic ß-sheet structures. The unexpected aggregation of NCBD needs to be taken into consideration given the multitude of protein-protein interactions and resulting biological functions mediated by CBP.


Assuntos
Amiloide/química , Amiloide/metabolismo , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/metabolismo , Agregados Proteicos , Agregação Patológica de Proteínas , Dobramento de Proteína , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
7.
Chem Sci ; 12(3): 1080-1089, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34163874

RESUMO

Intrinsically disordered proteins (IDPs) constitute a large portion of "Dark Proteome" - difficult to characterize or yet to be discovered protein structures. Here we used conformationally constrained α-methylated amino acids to bias the conformational ensemble in the free unstructured activation domain of transcriptional coactivator ACTR. Different sites and patterns of substitutions were enabled by chemical protein synthesis and led to distinct populations of α-helices. A specific substitution pattern resulted in a substantially higher binding affinity to nuclear coactivator binding domain (NCBD) of CREB-binding protein, a natural binding partner of ACTR. The first X-ray structure of the modified ACTR domain - NCBD complex visualized a unique conformation of ACTR and confirmed that the key α-methylated amino acids are localized within α-helices in the bound state. This study demonstrates a strategy for characterization of individual conformational states of IDPs.

9.
Angew Chem Int Ed Engl ; 58(43): 15324-15328, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31449707

RESUMO

Vibrational strong coupling (VSC) has recently emerged as a completely new tool for influencing chemical reactivity. It harnesses electromagnetic vacuum fluctuations through the creation of hybrid states of light and matter, called polaritonic states, in an optical cavity resonant to a molecular absorption band. Here, we investigate the effect of vibrational strong coupling of water on the enzymatic activity of pepsin, where a water molecule is directly involved in the enzyme's chemical mechanism. We observe an approximately 4.5-fold decrease of the apparent second-order rate constant kcat /Km when coupling the water stretching vibration, whereas no effect was detected for the strong coupling of the bending vibration. The possibility of modifying enzymatic activity by coupling water demonstrates the potential of VSC as a new tool to study biochemical reactivity.

10.
Chem Sci ; 9(25): 5594-5599, 2018 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-30061991

RESUMO

Discovering molecular probes that specifically recognize distinct amyloid structures is highly important for physiological studies of protein-misfolding diseases as well as for the development of diagnostic reagents and inhibitors of amyloid self-assembly. Here, we demonstrate an approach that allows for identification of N-methylated peptides that are specific binders for a particular amyloid fiber subtype (or polymorph). Protein design and chemical synthesis were used to produce covalently tethered amyloid analogues with molecular masses approaching 24 kDa and containing nine copies of an amyloidogenic peptide. Such engineered constructs served as a molecular testing platform to evaluate the aggregation properties and solubility as a function of N-methylation pattern. An advantage of the method is the possibility of biophysical characterization of amyloid constructs in solution.

11.
Chem Commun (Camb) ; 53(53): 7369-7372, 2017 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-28604862

RESUMO

Non-canonical α-methyl amino acids were incorporated at various sites in the sequence of intrinsically disordered activation domain from the p160 transcriptional co-activator (ACTR) to facilitate the formation of α-helical structures. Kinetic and thermodynamic data confirm the induced fit mechanism of complex formation between the synthesized ACTR variants and the nuclear co-activator binding domain (NCBD).


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/síntese química , Dobramento de Proteína , Aminoácidos/química , Cinética , Ligação Proteica , Conformação Proteica , Termodinâmica , Fatores de Transcrição/química
12.
J Phys Chem Lett ; 7(20): 4159-4164, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27689759

RESUMO

In quantum electrodynamics, matter can be hybridized to confined optical fields by a process known as light-matter strong coupling. This gives rise to new hybrid light-matter states and energy levels in the coupled material, leading to modified physical and chemical properties. Here, we report for the first time the strong coupling of vibrational modes of proteins with the vacuum field of a Fabry-Perot mid-infrared cavity. For two model systems, poly(l-glutamic acid) and bovine serum albumin, strong coupling is confirmed by the anticrossing in the dispersion curve, the square root dependence on the concentration, and a vacuum Rabi splitting that is larger than the cavity and vibration line widths. These results demonstrate that strong coupling can be applied to the study of proteins with many possible applications including the elucidation of the role of vibrational dynamics in enzyme catalysis and in H/D exchange experiments.

13.
Chembiochem ; 17(23): 2274-2285, 2016 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-27717158

RESUMO

Polymorphism is a common property of amyloid fibers that complicates their detailed structural and functional studies. Here we report experiments illustrating the chemical principles that enable the formation of amyloid polymorphs with distinct stoichiometric composition. Using appropriate covalent tethering we programmed self-assembly of a model peptide corresponding to the [20-41] fragment of human ß2-microglobulin into fibers with either trimeric or dimeric amyloid cores. Using a set of biophysical and biochemical methods we demonstrated their distinct structural, morphological, and templating properties. Furthermore, we showed that supramolecular approaches in which the peptide is modified with bulky substituents can also be applied to modulate the formation of different fiber polymorphs. Such strategies, when applied to disease-related peptides and proteins, will greatly help in the evaluation of the biological properties of structurally distinct amyloids.


Assuntos
Amiloide/química , Amiloide/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação Proteica
14.
J Pept Sci ; 22(5): 290-304, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26929241

RESUMO

Insoluble amyloid fibers represent a pathological signature of many human diseases. To treat such diseases, inhibition of amyloid formation has been proposed as a possible therapeutic strategy. d-Peptides, which possess high proteolytic stability and lessened immunogenicity, are attractive candidates in this context. However, a molecular understanding of chiral recognition phenomena for d-peptides and l-amyloids is currently incomplete. Here we report experiments on amyloid growth of individual enantiomers and their mixtures for two distinct polypeptide systems of different length and structural organization: a 44-residue covalently-linked dimer derived from a peptide corresponding to the [20-41]-fragment of human ß2-microglobulin (ß2m) and the 99-residue full-length protein. For the dimeric [20-41]ß2m construct, a combination of electron paramagnetic resonance of nitroxide-labeled constructs and (13) C-isotope edited FT-IR spectroscopy of (13) C-labeled preparations was used to show that racemic mixtures precipitate as intact homochiral fibers, i.e. undergo spontaneous Pasteur-like resolution into a mixture of left- and right-handed amyloids. In the case of full-length ß2m, the presence of the mirror-image d-protein affords morphologically distinct amyloids that are composed largely of enantiopure domains. Removal of the l-component from hybrid amyloids by proteolytic digestion results in their rapid transformation into characteristic long straight d-ß2m amyloids. Furthermore, the full-length d-enantiomer of ß2m was found to be an efficient inhibitor of l-ß2m amyloid growth. This observation highlights the potential of longer d-polypeptides for future development into inhibitors of amyloid propagation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Amiloide/química , Peptídeos/síntese química , Microglobulina beta-2/química , Humanos , Modelos Moleculares , Peptídeos/química , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo
15.
PLoS One ; 10(12): e0143948, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26650386

RESUMO

Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-ß-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.


Assuntos
Amiloide/química , Ensaios de Triagem em Larga Escala/métodos , Fragmentos de Peptídeos/química , Prebióticos , Catálise , Dicroísmo Circular , Humanos , Hidrólise , Microscopia Eletrônica de Transmissão , Espectroscopia de Infravermelho com Transformada de Fourier
16.
J Am Chem Soc ; 137(7): 2524-35, 2015 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-25633201

RESUMO

Conversion of soluble folded proteins into insoluble amyloids generally proceeds in three distinct mechanistic stages: (1) initial protein misfolding into aggregation-competent conformers, (2) subsequent formation of oligomeric species and, finally, (3) self-assembly into extended amyloid fibrils. In the work reported herein, we interrogated the amyloidogenesis mechanism of human ß2-microglobulin (ß2m), which is thought to be triggered by a pivotal cis-trans isomerization of a proline residue at position 32 in the polypeptide, with nonstandard amino acids. Using chemical protein synthesis we prepared a ß2m analogue in which Pro32 was replaced by the conformationally constrained amino acid α-methylproline (MePro). The strong propensity of MePro to adopt a trans prolyl bond led to enhanced population of a non-native [trans-MePro32]ß2m protein conformer, which readily formed oligomers at neutral pH. In the presence of the antibiotic rifamycin SV, which inhibits amyloid growth of wild-type ß2m, [MePro32]ß2m was nearly quantitatively converted into different spherical oligomeric species. Self-assembly into amyloid fibrils was not observed in the absence of seeding, however, even at low pH (<3), where wild-type ß2m spontaneously forms amyloids. Nevertheless, we found that aggregation-preorganized [MePro32]ß2m can act in a prion-like fashion, templating misfolded conformations in a natively folded protein. Overall, these results provide detailed insight into the role of cis-trans isomerization of Pro32 and ensuing structural rearrangements that lead to initial ß2m misfolding and aggregation. They corroborate the view that conformational protein dynamics enabled by reversible Pro32 cis-trans interconversion rather than simple population of the trans conformer is critical for both nucleation and subsequent growth of ß2m amyloid structures.


Assuntos
Substituição de Aminoácidos , Amiloide/química , Prolina/análogos & derivados , Multimerização Proteica , Microglobulina beta-2/química , Humanos , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Prolina/química , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica , Estrutura Secundária de Proteína , Rifamicinas/farmacologia , Estereoisomerismo , Microglobulina beta-2/genética
17.
Proc Natl Acad Sci U S A ; 110(50): 20051-6, 2013 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-24262149

RESUMO

The human protein ß2-microglobulin (ß2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger ß2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis. We show that ß2m's stability, (un)folding, and aggregation properties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the ß2m monomer was either stabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the stereoisomeric (2S,4R)-fluoroproline, which disfavors this conformation. However, substitution of Pro32 with 4,4-difluoroproline, which has nearly the same cis-trans preference as proline but an enhanced isomerization rate, caused pronounced destabilization of the protein and increased oligomerization at neutral pH. More remarkably, these subtle alterations in chemical composition--incorporation of one or two fluorine atoms into a single proline residue in the 99 amino acid long protein--modulated the aggregation properties of ß2m, inducing the formation of polymorphically distinct amyloid fibrils. These results highlight the importance of conformational dynamics for molecular assembly of an amyloid cross-ß structure and provide insights into mechanistic aspects of Pro32 cis-trans isomerism in ß2m aggregation.


Assuntos
Amidas/química , Amiloidose/prevenção & controle , Diálise/efeitos adversos , Modelos Moleculares , Prolina/química , Conformação Proteica , Microglobulina beta-2/biossíntese , Amiloidose/etiologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Isomerismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Solubilidade , Microglobulina beta-2/química
18.
Org Biomol Chem ; 10(30): 5887-91, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22659831

RESUMO

Total chemical synthesis was used to site-specifically (13)C-label active site Asp25 and Asp25' residues in HIV-1 protease and in several chemically synthesized analogues of the enzyme molecule. (13)C NMR measurements were consistent with a monoprotonated state for the catalytic dyad formed by the interacting Asp25, Asp25' side chain carboxyls.


Assuntos
Ácido Aspártico , Biocatálise , Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/enzimologia , Sítios de Ligação , Isótopos de Carbono , Técnicas de Química Sintética , Marcação por Isótopo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Especificidade por Substrato
20.
Proc Natl Acad Sci U S A ; 108(52): 20982-7, 2011 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-22158985

RESUMO

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.


Assuntos
Protease de HIV/química , Protease de HIV/síntese química , Modelos Moleculares , Conformação Proteica , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular
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