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1.
Opt Lett ; 47(10): 2538-2541, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35561395

RESUMO

In fluorescence diffuse optical tomography (fDOT), the quality of reconstruction is severely limited by mismodeling and ill-posedness of inverse problems. Although data-driven deep learning methods improve the quality of image reconstruction, the network architecture lacks interpretability and requires a lot of data for training. We propose an interpretable model-driven projected gradient descent network (MPGD-Net) to improve the quality of fDOT reconstruction using only a few training samples. MPGD-Net unfolds projected gradient descent into a novel deep network architecture that is naturally interpretable. Simulation and in vivo experiments show that MPGD-Net greatly improves the fDOT reconstruction quality with superior generalization ability.

2.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
3.
Nat Commun ; 11(1): 5096, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037221

RESUMO

Folding of individual domains in large proteins during translation helps to avoid otherwise prevalent inter-domain misfolding. How folding intermediates observed in vitro for the majority of proteins relate to co-translational folding remains unclear. Combining in vivo and single-molecule experiments, we followed the co-translational folding of the G-domain, encompassing the first 293 amino acids of elongation factor G. Surprisingly, the domain remains unfolded until it is fully synthesized, without collapsing into molten globule-like states or forming stable intermediates. Upon fully emerging from the ribosome, the G-domain transitions to its stable native structure via folding intermediates. Our results suggest a strictly sequential folding pathway initiating from the C-terminus. Folding and synthesis thus proceed in opposite directions. The folding mechanism is likely imposed by the final structure and might have evolved to ensure efficient, timely folding of a highly abundant and essential protein.


Assuntos
Fator G para Elongação de Peptídeos/biossíntese , Fator G para Elongação de Peptídeos/química , Dobramento de Proteína , Luminescência , Fator G para Elongação de Peptídeos/genética , Biossíntese de Proteínas , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Imagem Individual de Molécula
4.
Proc Natl Acad Sci U S A ; 116(51): 25641-25648, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776255

RESUMO

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.


Assuntos
Biossíntese de Proteínas/fisiologia , Domínios Proteicos/fisiologia , Dobramento de Proteína , Proteínas , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Pinças Ópticas , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Ribossomos , Imagem Individual de Molécula , Termodinâmica
5.
Opt Lett ; 44(13): 3222-3225, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31259926

RESUMO

We present a method to accelerate image reconstruction in fluorescence molecular tomography based on the historical path fluorescence Monte Carlo model. The method exploits a first-order approximation expression during the fluorescence excitation-transmission process to merge the path and state information of the photon in a voxel. The experiments show that our method not only greatly reduces the amount of data required for storage in the hard disk and accelerates image reconstruction, but also maintains the quantitative and positioning accuracy of the conventional method.


Assuntos
Fluorescência , Processamento de Imagem Assistida por Computador/métodos , Tomografia , Fótons , Fatores de Tempo
6.
Mol Cell ; 74(2): 310-319.e7, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30852061

RESUMO

Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding.


Assuntos
Fator G para Elongação de Peptídeos/química , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Pinças Ópticas , Fator G para Elongação de Peptídeos/genética , Peptídeos/química , Peptídeos/genética , Domínios Proteicos/genética , Proteoma/química , Proteoma/genética , Ribossomos/química , Ribossomos/genética
7.
J Mol Biol ; 430(22): 4580-4591, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29981746

RESUMO

All cellular proteins are synthesized by the ribosome, an intricate molecular machine that translates the information of protein coding genes into the amino acid alphabet. The linear polypeptides synthesized by the ribosome must generally fold into specific three-dimensional structures to become biologically active. Folding has long been recognized to begin before synthesis is complete. Recently, biochemical and biophysical studies have shed light onto how the ribosome shapes the folding pathways of nascent proteins. Here, we discuss recent progress that is beginning to define the role of the ribosome in the folding of newly synthesized polypeptides.


Assuntos
Proteínas/química , Proteínas/metabolismo , Ribossomos/metabolismo , Animais , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Espectrometria de Fluorescência
9.
Elife ; 62017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29022880

RESUMO

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here, we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of 'energetic frustration'. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.


Assuntos
Regulação Alostérica , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Isoformas de Proteínas/química , Receptores de Glucocorticoides/química , Fatores de Transcrição/química , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Conformação Proteica , Isoformas de Proteínas/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo
10.
Protein Sci ; 26(7): 1439-1451, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28474852

RESUMO

Correct folding is a prerequisite for the biological activity of most proteins. Folding has largely been studied using in vitro refolding assays with isolated small, robustly folding proteins. A substantial fraction of all cellular proteomes is composed of multidomain proteins that are often not amenable to this approach, and their folding remains poorly understood. These large proteins likely begin to fold during their synthesis by the ribosome, a large molecular machine that translates the genetic code. The ribosome affects how folding proceeds, but the underlying mechanisms remain largely obscure. We have utilized optical tweezers to study the folding of elongation factor G, a multidomain protein composed of five domains. We find that interactions among unfolded domains interfere with productive folding in the full-length protein. The N-terminal G-domain constitutes an independently folding unit that, upon in vitro refolding, adopts two similar states that correspond to the natively folded and a non-native, possibly misfolded structure. The ribosome destabilizes both of these states, suggesting a mechanism by which terminal misfolding into highly stable, non-native structures is avoided. The ribosome may thus directly contribute to efficient folding by modulating the folding of nascent multidomain proteins.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/fisiologia , Dobramento de Proteína , Ribossomos/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/genética , Domínios Proteicos , Ribossomos/química , Ribossomos/genética
11.
J Biol Chem ; 287(12): 9137-46, 2012 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-22262834

RESUMO

Rtt107 (regulator of Ty1 transposition 107; Esc4) is a DNA repair protein from Saccharomyces cerevisiae that can restore stalled replication forks following DNA damage. There are six BRCT (BRCA1 C-terminal) domains in Rtt107 that act as binding sites for other recruited proteins during DNA repair. Several Rtt107 binding partners have been identified, including Slx4, Rtt101, Rad55, and the Smc5/6 (structural maintenance of chromosome) protein complex. Rtt107 can reportedly be recruited to chromatin in the presence of Rtt101 and Rtt109 upon DNA damage, but the chromatin-binding site of Rtt107 has not been identified. Here, we report our investigation of the interaction between phosphorylated histone H2A (γH2A) and the C-terminal tandem BRCT repeats (BRCT(5)-BRCT(6)) of Rtt107. The crystal structures of BRCT(5)-BRCT(6) alone and in a complex with γH2A reveal the molecular basis of the Rtt107-γH2A interaction. We used in vitro mutagenesis and a fluorescence polarization assay to confirm the location of the Rtt107 motif that is crucial for this interaction. In addition, these assays indicated that this interaction requires the phosphorylation of H2A. An in vivo phenotypic analysis in yeast demonstrated the critical role of BRCT(5)-BRCT(6) and its interaction with γH2A during the DNA damage response. Our results shed new light on the molecular mechanism by which Rtt107 is recruited to chromatin in response to stalled DNA replication forks.


Assuntos
Reparo do DNA , Histonas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequências de Repetição em Tandem , Motivos de Aminoácidos , Sequência de Aminoácidos , Cromatina/genética , Cromatina/metabolismo , Cristalografia por Raios X , Dano ao DNA , Histonas/química , Histonas/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
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