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1.
Chem Rev ; 122(10): 9795-9847, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35446555

RESUMO

Solid-state NMR spectroscopy (ssNMR) with magic-angle spinning (MAS) enables the investigation of biological systems within their native context, such as lipid membranes, viral capsid assemblies, and cells. However, such ambitious investigations often suffer from low sensitivity due to the presence of significant amounts of other molecular species, which reduces the effective concentration of the biomolecule or interaction of interest. Certain investigations requiring the detection of very low concentration species remain unfeasible even with increasing experimental time for signal averaging. By applying dynamic nuclear polarization (DNP) to overcome the sensitivity challenge, the experimental time required can be reduced by orders of magnitude, broadening the feasible scope of applications for biological solid-state NMR. In this review, we outline strategies commonly adopted for biological applications of DNP, indicate ongoing challenges, and present a comprehensive overview of biological investigations where MAS-DNP has led to unique insights.


Assuntos
Núcleo Celular , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos
2.
Angew Chem Int Ed Engl ; 59(29): 11937-11942, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32219972

RESUMO

Alkaptonuria (AKU) is a rare disease characterized by high levels of homogentisic acid (HGA); patients suffer from tissue ochronosis: dark brown pigmentation, especially of joint cartilage, leading to severe early osteoarthropathy. No molecular mechanism links elevated HGA to ochronosis; the pigment's chemical identity is still not known, nor how it induces joint cartilage degradation. Here we give key insight on HGA-derived pigment composition and collagen disruption in AKU cartilage. Synthetic pigment and pigmented human cartilage tissue both showed hydroquinone-resembling NMR signals. EPR spectroscopy showed that the synthetic pigment contains radicals. Moreover, we observed intrastrand disruption of collagen triple helix in pigmented AKU human cartilage, and in cartilage from patients with osteoarthritis. We propose that collagen degradation can occur via transient glycyl radicals, the formation of which is enhanced in AKU due to the redox environment generated by pigmentation.


Assuntos
Alcaptonúria/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Pigmentação , Espectroscopia de Ressonância de Spin Eletrônica , Ácido Homogentísico/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Oxirredução , Pigmentos Biológicos/química
3.
J Biomol NMR ; 66(2): 93-98, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27699524

RESUMO

We have prepared mouse fur extensively 13C,15N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. 13C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and ß-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.


Assuntos
Pelo Animal/química , Queratinas/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Aminoácidos , Animais , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Ressonância Magnética Nuclear Biomolecular
4.
J Biomol NMR ; 63(2): 119-23, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26407607

RESUMO

An appreciable level of isotope labelling is essential for future NMR structure elucidation of mammalian biomaterials, which are either poorly expressed, or unexpressable, using micro-organisms. We present a detailed protocol for high level (13)C enrichment even in slow turnover murine biomaterials (fur keratin), using a customized diet supplemented with commercial labelled algal hydrolysate and formulated as a gel to minimize wastage, which female mice consumed during pregnancy and lactation. This procedure produced approximately eightfold higher fur keratin labelling in pups, exposed in utero and throughout life to label, than in adults exposed for the same period, showing both the effectiveness, and necessity, of this approach.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Animais , Camundongos , Ressonância Magnética Nuclear Biomolecular/métodos , Especificidade de Órgãos
5.
J Inherit Metab Dis ; 34(6): 1137-40, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21735270

RESUMO

UNLABELLED: In pilot studies of the usefulness of solid state nuclear magnetic resonance spectroscopy in characterizing chemical and molecular structural effects of alkaptonuria on connective tissue, we have obtained (13) C spectra from articular cartilage from an AKU patient. An apparently normal anatomical location yielded a cross polarization magic angle spinning spectrum resembling literature spectra and dominated by collagen and glycosaminoglycan signals. All spectral linewidths from strongly pigmented ochronotic cartilage however were considerably increased relative to the control indicating a marked increase in collagen molecular disorder. This disordering of cartilage structural protein parallels, at the atomic level, the disordering revealed at higher length scales by microscopy. We also demonstrate that the abnormal spectra from ochronotic cartilage fit with the abnormality in the structure of collagen fibres at the ultrastructural level, whereby large ochronotic deposits appear to alter the structure of the collagen fibre by invasion and cross linking. SUMMARY: Increased signal linewidths in solid state NMR spectra of ochronotic articular cartilage from an AKU patient relative to linewidths in normal, control, cartilage reveals a marked decrease in collagen molecular order in the diseased tissue. This atomic level disordering parallels higher length scale disorder revealed by microscopic techniques.


Assuntos
Alcaptonúria/complicações , Doenças das Cartilagens/patologia , Cartilagem Articular/química , Colágeno/análise , Colágeno/ultraestrutura , Glicosaminoglicanos/análise , Ocronose/diagnóstico por imagem , Idoso , Doenças das Cartilagens/etiologia , Cartilagem Articular/ultraestrutura , Colágeno/química , Feminino , Glicosaminoglicanos/química , Humanos , Espectroscopia de Ressonância Magnética/métodos , Ocronose/etiologia , Projetos Piloto , Ultrassonografia
6.
Adv Mater ; 32(9): e1903942, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31984551

RESUMO

Poly(heptazine imides) hosting cobalt ions as countercations are presented as promising electrocatalysts for the oxygen evolution reaction (OER). A facile mixed-salt melt-assisted condensation is developed to prepare such cobalt poly(heptazine imides) (PHI-Co). The Co ions can be introduced in well-controlled amounts using this method, and are shown to be atomically dispersed within the imide-linked heptazine matrix. When applied to electrocatalytic OER, PHI-Co shows a remarkable activity with an overpotential of 324 mV and Tafel slope of 44 mV dec-1 in 1 m KOH.

7.
Methods Mol Biol ; 1944: 57-77, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840235

RESUMO

Solid-state nuclear magnetic resonance spectroscopy (ssNMR) is an emerging technique in structural methods of studying collagen proteins, capable of identifying features on an atomic length scale in tissues and protein samples without extensive extraction or purification. Hydroxylation is a key posttranslational modification of collagen that gives rise to distinctive signals in the ssNMR spectrum of collagen proteins. Here we outline the type of information that ssNMR can provide and describe the procedures involved in a ssNMR structural study, with particular focus on using dynamic nuclear polarization to enhance sensitivity for detecting hydroxylysine residues by ssNMR.


Assuntos
Osso e Ossos/metabolismo , Colágeno/química , Colágeno/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Processamento de Proteína Pós-Traducional , Pele/metabolismo , Animais , Humanos , Hidroxilação , Camundongos
8.
RSC Adv ; 9(46): 26686-26690, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-35528564

RESUMO

Sensitivity enhancement by isotope enrichment and DNP NMR enables detection of minor but biologically relevant species in native intact bone, including nucleic acids, choline from phospholipid headgroups, and histidinyl and hydroxylysyl groups. Labelled matrix from the aggressive osteosarcoma K7M2 cell line confirms the assignments of nucleic acid signals arising from purine, pyrimidine, ribose, and deoxyribose species. Detection of these species is an important and necessary step in elucidating the atomic level structural basis of their functions in intact tissue.

9.
Cell Rep ; 27(11): 3124-3138.e13, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31189100

RESUMO

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.


Assuntos
Biomineralização , Dano ao DNA , Poli Adenosina Difosfato Ribose/metabolismo , Calcificação Vascular/metabolismo , Adolescente , Adulto , Idoso , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Bovinos , Linhagem Celular , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoblastos/metabolismo , Osteoblastos/patologia , Estresse Oxidativo , Ratos , Ratos Wistar , Ovinos
10.
Chem Commun (Camb) ; 54(89): 12570-12573, 2018 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-30299444

RESUMO

The sparse but functionally essential post-translational collagen modification 5-hydroxylysine can undergo further transformations, including crosslinking, O-glycosylation, and glycation. Dynamic nuclear polarization (DNP) and stable isotope enriched lysine incorporation provide sufficient solid-state NMR sensitivity to identify these adducts directly in skin and vascular smooth muscle cell extracellular matrix (ECM), without extraction procedures, by comparison with chemical shifts of model compounds. Thus, DNP provides access to the elucidation of structural consequences of collagen modifications in intact tissue.

11.
Sci Rep ; 8(1): 13809, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218106

RESUMO

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional 13C-13C correlation NMR spectroscopy on 13C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils. We propose that the Gly-Pro-Hyp triplets in fibrillar collagens provide fibril "expansion joints" to maintain molecular ordering within the fibril, thereby preserving the structural integrity of ligand binding sites.


Assuntos
Colágeno/química , Colágeno/metabolismo , Prolina/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Feminino , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/fisiologia , Glicina/química , Hidroxiprolina/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Peptídeos/química , Prolina/fisiologia , Conformação Proteica , Ovinos
12.
J Colloid Interface Sci ; 350(1): 205-11, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20638074

RESUMO

Quantitative analysis of a number of potential fluorous surfactants, prepared with a view to stabilisation of microdroplets in microfluidic systems is presented. The surfactants tested comprised compounds with both perfluoropolyether (PFPE) and perfluoroalkyl (PFA) tails, along with three classes of hydrophilic head group, including crown ethers and hexaethylene gylcol. Surfactants were tested for activity using the pendant drop technique. Six compounds proved highly effective and efficient surfactants, with gamma(CMC)<10 mN/m and CMCs in the sub-millimolar range. These six compounds stabilised aqueous microdroplets in fluorous oils within poly(dimethylsiloxane) (PDMS) microdevices to a greater degree than commonly used pseudosurfactants such as perfluorooctanol.

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