Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Phys Chem Lett ; 12(32): 7777-7782, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34374547

RESUMO

Enzyme catalysis achieves tremendous rate accelerations. Enzyme reaction centers provide a constraint geometry that preferentially binds an activated form of the substrate and thus lowers the energy barrier. However, this transition state picture neglects the flexibility of proteins and its role in enzymatic catalysis. Especially for proton transfer reactions, it has been suggested that motions of the protein modulate the donor-acceptor distance and prepare a tunneling-ready state. We report the detection of frequency fluctuations of an azide anion (N3-) bound in the active site of the protein carbonic anhydrase II, where a low-frequency mode of the protein has been proposed to facilitate proton transfer over two water molecules during the catalyzed reaction. 2D-IR spectroscopy resolves an underdamped low-frequency mode at about 1 THz (30 cm-1). We find its frequency to be viscosity- and temperature-dependent and to decrease by 6 cm-1 between 230 and 320 K, reporting the softening of the mode's potential.


Assuntos
Anidrase Carbônica II/química , Animais , Azidas/química , Domínio Catalítico , Bovinos , Prótons , Espectrofotometria Infravermelho/métodos , Temperatura , Vibração , Viscosidade , Água/química
2.
Phys Chem Chem Phys ; 22(40): 22963-22972, 2020 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-33029608

RESUMO

Incorporation of minimally perturbative vibrational probes into proteins allows combination of the femtosecond time resolution of two dimensional infrared (2D-IR) spectroscopy with a spatial resolution on the level of single side chains. Here, we apply the thiocyanate (-SCN) label introduced by the cyanylation of cysteine to probe local dynamics in the photo-switchable protein PYP. We incorporated the -SCN label into five positions of the protein structure including PYP's core region, its solvent exposed surface and the chromophore-binding pocket. The analysis of -SCN's time dependent 2D-IR lineshape provides insight into the timescales and amplitudes of the dynamics in the label's protein and solvent microenvironment. We present a detailed analysis of the local protein dynamics found at all five labelling positions in PYP's dark state (pG). Absorption of a blue photon triggers the isomerisation of PYP's chromophore and eventually leads to an overall reorganisation of the protein structure, where PYP ends up in a less structured signalling state pB. Employing 2D-IR spectroscopy also on the signalling state allows assessment of the change of local dynamics compared to the pG state.

3.
Phys Chem Chem Phys ; 22(10): 5463-5475, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32096510

RESUMO

The calcium sensor protein calmodulin is ubiquitous among eukaryotes. It translates intracellular Ca2+ influx (by a decrease of conformational flexibility) into increased target recognition affinity. Here we demonstrate that by using the IR reporter -SCN in combination with 2D-IR spectroscopy, global structure changes and local dynamics, degree of solvent exposure and protein-ligand interaction can be characterised in great detail. The long vibrational lifetime of the -SCN label allows for centerline slope analysis of the 2D-IR line shape up to 120 ps to deduce the frequency-frequency correlation function (FFCF) of the -SCN label in various states and label positions in the protein. Based on that we show clear differences between a solvent exposed site, the environment close to the Ca2+ binding motif and three highly conserved positions for ligand binding. Furthermore, we demonstrate how these dynamics are affected by conformational change induced by the addition of Ca2+ ions and by interaction with a short helical peptide mimicking protein binding. We show that the binding mode is strongly heterogeneous among the probed key binding methionine residues. SCN's vibrational relaxation is dominated by intermolecular contributions. Changes in the vibrational lifetime upon changing between H2O and D2O buffer therefore provide a robust measure for water accessibility of the label. Characterising -SCN's extinction coefficient, vibrational lifetime in light and heavy water and its FFCF we demonstrate the vast potential it has as a label especially for nonlinear spectroscopies, such as 2D-IR spectroscopy.


Assuntos
Calmodulina/química , Espectrofotometria Infravermelho , Calmodulina/metabolismo , Óxido de Deutério/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Solventes/química , Vibração , Água/química
4.
Anal Chem ; 92(1): 1024-1032, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31769286

RESUMO

The application of vibrational labels such as thiocyanate  (-S-C≡N) for studying protein structure and dynamics is thriving. Absorption spectroscopy is usually employed to obtain wavenumber and line shape of the label. An observable of great significance might be the vibrational lifetime, which can be obtained by pump probe or 2D-IR spectroscopy. Due to the insulating effect of the heavy sulfur atom in the case of the SCN label, the lifetime of the C≡N oscillator is expected to be particularly sensitive to its surrounding as it is not dominated by through-bond relaxation. We therefore investigate the vibrational lifetime of the SCN label at various positions in the blue light sensor protein Photoactive Yellow Protein (PYP) in the ground state and signaling state of the photoreceptor. We find that the vibrational lifetime of the C≡N stretching mode is strongly affected both by its protein environment and by the degree of exposure to the solvent. Even for label positions where the line shape and wavenumber observed by FTIR are barely changing upon activation of the photoreceptor, we find that the lifetime can change considerably. To obtain an unambiguous measure for the solvent exposure of the labeled site, we show that it is imperative to compare the lifetimes in H2O and D2O. Importantly, the lifetimes shorten in H2O as compared to D2O for water exposed labels, while they stay largely the same for buried labels. We quantify this effect by defining a solvent exclusion coefficient (SEC). The response of the label's vibrational lifetime to its solvent exposure renders it a suitable universal probe for protein investigations. This applies even to systems that are otherwise hard to address, such as transient or short-lived states, which could be created during a protein's working cycle (as here in PYP) or during protein folding. It is also applicable to flexible systems (intrinsically disordered proteins), protein-protein and protein-membrane interactions.


Assuntos
Proteínas de Bactérias/química , Óxido de Deutério/química , Fotorreceptores Microbianos/química , Tiocianatos/química , Proteínas de Bactérias/efeitos da radiação , Halorhodospira halophila/química , Luz , Simulação de Dinâmica Molecular , Fotorreceptores Microbianos/efeitos da radiação , Conformação Proteica , Espectrofotometria Infravermelho , Tiocianatos/efeitos da radiação , Vibração
5.
Proc Natl Acad Sci U S A ; 115(39): 9744-9749, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30201720

RESUMO

Cotranslational folding (CTF) is a fundamental molecular process that ensures efficient protein biosynthesis and minimizes the formation of misfolded states. However, the complexity of this process makes it extremely challenging to obtain structural characterizations of CTF pathways. Here, we correlate observations of translationally arrested nascent chains with those of a systematic C-terminal truncation strategy. We create a detailed description of chain length-dependent free energy landscapes associated with folding of the FLN5 filamin domain, in isolation and on the ribosome, and thus, quantify a substantial destabilization of the native structure on the ribosome. We identify and characterize two folding intermediates formed in isolation, including a partially folded intermediate associated with the isomerization of a conserved cis proline residue. The slow folding associated with this process raises the prospect that neighboring unfolded domains might accumulate and misfold during biosynthesis. We develop a simple model to quantify the risk of misfolding in this situation and show that catalysis of folding by peptidyl-prolyl isomerases is sufficient to eliminate this hazard.


Assuntos
Filaminas/biossíntese , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dobramento de Proteína , Modificação Traducional de Proteínas , Deficiências na Proteostase/metabolismo , Ribossomos/metabolismo , Sequências de Repetição em Tandem , Termodinâmica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...