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1.
Nat Commun ; 10(1): 4598, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31601802

RESUMO

mRNA contexts containing a 'slippery' sequence and a downstream secondary structure element stall the progression of the ribosome along the mRNA and induce its movement into the -1 reading frame. In this study we build a thermodynamic model based on Bayesian statistics to explain how -1 programmed ribosome frameshifting can work. As training sets for the model, we measured frameshifting efficiencies on 64 dnaX mRNA sequence variants in vitro and also used 21 published in vivo efficiencies. With the obtained free-energy difference between mRNA-tRNA base pairs in the 0 and -1 frames, the frameshifting efficiency of a given sequence can be reproduced and predicted from the tRNA-mRNA base pairing in the two frames. Our results further explain how modifications in the tRNA anticodon modulate frameshifting and show how the ribosome tunes the strength of the base-pair interactions.

2.
J Chem Inf Model ; 59(10): 4093-4099, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31525920

RESUMO

Given the need for modern researchers to produce open, reproducible scientific output, the lack of standards and best practices for sharing data and workflows used to produce and analyze molecular dynamics (MD) simulations has become an important issue in the field. There are now multiple well-established packages to perform molecular dynamics simulations, often highly tuned for exploiting specific classes of hardware, each with strong communities surrounding them, but with very limited interoperability/transferability options. Thus, the choice of the software package often dictates the workflow for both simulation production and analysis. The level of detail in documenting the workflows and analysis code varies greatly in published work, hindering reproducibility of the reported results and the ability for other researchers to build on these studies. An increasing number of researchers are motivated to make their data available, but many challenges remain in order to effectively share and reuse simulation data. To discuss these and other issues related to best practices in the field in general, we organized a workshop in November 2018 ( https://bioexcel.eu/events/workshop-on-sharing-data-from-molecular-simulations/ ). Here, we present a brief overview of this workshop and topics discussed. We hope this effort will spark further conversation in the MD community to pave the way toward more open, interoperable, and reproducible outputs coming from research studies using MD simulations.

3.
J Chem Theory Comput ; 15(9): 5087-5102, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31402660

RESUMO

Circular dichroism (CD) spectroscopy is a highly sensitive but low-resolution technique to study the structure of proteins. Combined with molecular modeling or other complementary techniques, CD spectroscopy can provide essential information at higher resolution. To this end, we introduce a new computational method to calculate the electronic circular dichroism spectra of proteins from a structural model or ensemble using the average secondary structure composition and a precalculated set of basis spectra. The method is designed for model validation to estimate the error of a given protein structural model based on the measured CD spectrum. We compared the predictive power of our method to that of existing algorithms, namely, DichroCalc and PDB2CD, and found that it predicts CD spectra more accurately. Our results indicate that the derived basis sets are robust to both experimental errors in the reference spectra and the choice of the secondary structure classification algorithm. For over 80% of the globular reference proteins, our basis sets accurately predict the experimental spectrum solely from their secondary structure composition. For the remaining 20%, correcting for intensity normalization considerably improves the prediction power. Additionally, we show that the predictions for short peptides and an example complex of intrinsically disordered proteins strongly benefit from accounting for side-chain contributions and structural flexibility.


Assuntos
Dicroísmo Circular , Proteínas/química , Algoritmos , Modelos Moleculares , Conformação Proteica , Teoria Quântica
4.
J Comput Chem ; 40(27): 2418-2431, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260119

RESUMO

We identify hardware that is optimal to produce molecular dynamics (MD) trajectories on Linux compute clusters with the GROMACS 2018 simulation package. Therefore, we benchmark the GROMACS performance on a diverse set of compute nodes and relate it to the costs of the nodes, which may include their lifetime costs for energy and cooling. In agreement with our earlier investigation using GROMACS 4.6 on hardware of 2014, the performance to price ratio of consumer GPU nodes is considerably higher than that of CPU nodes. However, with GROMACS 2018, the optimal CPU to GPU processing power balance has shifted even more toward the GPU. Hence, nodes optimized for GROMACS 2018 and later versions enable a significantly higher performance to price ratio than nodes optimized for older GROMACS versions. Moreover, the shift toward GPU processing allows to cheaply upgrade old nodes with recent GPUs, yielding essentially the same performance as comparable brand-new hardware. © 2019 Wiley Periodicals, Inc.

5.
Proc Natl Acad Sci U S A ; 116(14): 6594-6601, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30890636

RESUMO

Receptor-ligand interactions are essential for biological function and their binding strength is commonly explained in terms of static lock-and-key models based on molecular complementarity. However, detailed information on the full unbinding pathway is often lacking due, in part, to the static nature of atomic structures and ensemble averaging inherent to bulk biophysics approaches. Here we combine molecular dynamics and high-speed force spectroscopy on the streptavidin-biotin complex to determine the binding strength and unbinding pathways over the widest dynamic range. Experiment and simulation show excellent agreement at overlapping velocities and provided evidence of the unbinding mechanisms. During unbinding, biotin crosses multiple energy barriers and visits various intermediate states far from the binding pocket, while streptavidin undergoes transient induced fits, all varying with loading rate. This multistate process slows down the transition to the unbound state and favors rebinding, thus explaining the long lifetime of the complex. We provide an atomistic, dynamic picture of the unbinding process, replacing a simple two-state picture with one that involves many routes to the lock and rate-dependent induced-fit motions for intermediates, which might be relevant for other receptor-ligand bonds.


Assuntos
Biotina/química , Modelos Químicos , Simulação de Dinâmica Molecular , Estreptavidina/química , Ligação Proteica
6.
Elife ; 82019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30829573

RESUMO

We present a correlation-driven molecular dynamics (CDMD) method for automated refinement of atomistic models into cryo-electron microscopy (cryo-EM) maps at resolutions ranging from near-atomic to subnanometer. It utilizes a chemically accurate force field and thermodynamic sampling to improve the real-space correlation between the modeled structure and the cryo-EM map. Our framework employs a gradual increase in resolution and map-model agreement as well as simulated annealing, and allows fully automated refinement without manual intervention or any additional rotamer- and backbone-specific restraints. Using multiple challenging systems covering a wide range of map resolutions, system sizes, starting model geometries and distances from the target state, we assess the quality of generated models in terms of both model accuracy and potential of overfitting. To provide an objective comparison, we apply several well-established methods across all examples and demonstrate that CDMD performs best in most cases.

7.
Chemphyschem ; 19(19): 2507-2511, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30047198

RESUMO

In the absence of a stable fold, transient secondary structure kinetics define the native state of the prototypical and pharmacologically relevant intrinsically disordered protein (IDP) α-Synuclein (aS). Here, we investigate kinetics preventing ordering and possibly pathogenic ß-sheet aggregation. Interestingly, transient ß-sheets form frequently at sub µs time scales precisely at the positions observed in aS amyloid fibrils. The formation kinetics competes with rapid secondary structure dissociation rates, thus explaining the low secondary structure content. The fast secondary structure dissociation times are very similar to the dynamics of tertiary structure rearrangements. These findings suggest that the fast dissociation kinetics slows down conformational selection processes for aS aggregation, which may be a general mechanism controlling the aggregation kinetics of IDPs.

8.
Nat Commun ; 9(1): 2375, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29915244

RESUMO

Scattering experiments with femtosecond high-intensity free-electron laser pulses provide a new route to macromolecular structure determination. While currently limited to nano-crystals or virus particles, the ultimate goal is scattering on single biomolecules. The main challenges in these experiments are the extremely low signal-to-noise ratio due to the very low expected photon count per scattering image, often well below 100, as well as the random orientation of the molecule in each shot. Here we present a de novo correlation-based approach and show that three coherently scattered photons per image suffice for structure determination. Using synthetic scattering data of a small protein, we demonstrate near-atomic resolution of 3.3 Å using 3.3 × 1010 coherently scattered photons from 3.3 × 109 images, which is within experimental reach. Further, our three-photon correlation approach is robust to additional noise from incoherent scattering; the number of disordered solvent molecules attached to the macromolecular surface should be kept small.

9.
Elife ; 72018 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-29652248

RESUMO

Microtubules (MTs) are key components of the cytoskeleton and play a central role in cell division and development. MT assembly is known to be associated with a structural change in [Formula: see text]-tubulin dimers from kinked to straight conformations. How GTP binding renders individual dimers polymerization-competent, however, is still unclear. Here, we have characterized the conformational dynamics and energetics of unassembled tubulin using atomistic molecular dynamics and free energy calculations. Contrary to existing allosteric and lattice models, we find that GTP-tubulin favors a broad range of almost isoenergetic curvatures, whereas GDP-tubulin has a much lower bending flexibility. Moreover, irrespective of the bound nucleotide and curvature, two conformational states exist differing in location of the anchor point connecting the monomers that affects tubulin bending, with one state being strongly favored in solution. Our findings suggest a new combined model in which MTs incorporate and stabilize flexible GTP-dimers with a specific anchor point state.

10.
Curr Opin Struct Biol ; 49: 27-35, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29202442

RESUMO

The ribosome is a macromolecular complex which is responsible for protein synthesis in all living cells according to their transcribed genetic information. Using X-ray crystallography and, more recently, cryo-electron microscopy (cryo-EM), the structure of the ribosome was resolved at atomic resolution in many functional and conformational states. Molecular dynamics simulations have added information on dynamics and energetics to the available structural information, thereby have bridged the gap to the kinetics obtained from single-molecule and bulk experiments. Here, we review recent computational studies that brought notable insights into ribosomal structure and function.

11.
Mol Cell ; 68(3): 515-527.e6, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100052

RESUMO

Ribosomes synthesizing proteins containing consecutive proline residues become stalled and require rescue via the action of uniquely modified translation elongation factors, EF-P in bacteria, or archaeal/eukaryotic a/eIF5A. To date, no structures exist of EF-P or eIF5A in complex with translating ribosomes stalled at polyproline stretches, and thus structural insight into how EF-P/eIF5A rescue these arrested ribosomes has been lacking. Here we present cryo-EM structures of ribosomes stalled on proline stretches, without and with modified EF-P. The structures suggest that the favored conformation of the polyproline-containing nascent chain is incompatible with the peptide exit tunnel of the ribosome and leads to destabilization of the peptidyl-tRNA. Binding of EF-P stabilizes the P-site tRNA, particularly via interactions between its modification and the CCA end, thereby enforcing an alternative conformation of the polyproline-containing nascent chain, which allows a favorable substrate geometry for peptide bond formation.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Peptídeos/metabolismo , Ribossomos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/ultraestrutura , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Peptídeos/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Ribossomos/ultraestrutura , Relação Estrutura-Atividade
12.
J Phys Chem Lett ; 8(16): 3759-3764, 2017 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-28742347

RESUMO

Hydration shells around ions are crucial for many fundamental biological and chemical processes. Their local physicochemical properties are quite different from those of bulk water and hard to probe experimentally. We address this problem by combining soft X-ray spectroscopy using a liquid jet and molecular dynamics (MD) simulations together with ab initio electronic structure calculations to elucidate the water-ion interaction in a MgCl2 solution at the molecular level. Our results reveal that salt ions mainly affect the electronic properties of water molecules in close vicinity and that the oxygen K-edge X-ray emission spectrum of water molecules in the first solvation shell differs significantly from that of bulk water. Ion-specific effects are identified by fingerprint features in the water X-ray emission spectra. While Mg2+ ions cause a bathochromic shift of the water lone pair orbital, the 3p orbital of the Cl- ions causes an additional peak in the water emission spectrum at around 528 eV.

13.
J Am Chem Soc ; 139(11): 4025-4034, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28253614

RESUMO

F1-ATPase is a highly efficient molecular motor that can synthesize ATP driven by a mechanical torque. Its ability to function reversibly in either direction requires tight mechanochemical coupling between the catalytic domain and the rotating central shaft, as well as temporal control of substrate binding and product release. Despite great efforts and significant progress, the molecular details of this synchronized and fine-tuned energy conversion mechanism are not fully understood. Here, we use extensive molecular dynamics simulations to reconcile recent single-molecule experiments with structural data and provide a consistent thermodynamic, kinetic and mechanistic description of the main rotary substep in the synthetic cycle of mammalian ATP synthase. The calculated free energy profiles capture a discrete pattern in the rotation of the central γ-shaft, with a metastable intermediate located-consistently with recent experimental findings-at 70° relative to the X-ray position. We identify this rotary step as the ATP-dependent substep, and find that the associated free energy input supports the mechanism involving concurrent nucleotide binding and release. During the main substep, our simulations show no significant opening of the ATP-bound ß subunit; instead, we observe that mechanical energy is transmitted to its nucleotide binding site, thus lowering the affinity for ATP. Simultaneously, the empty subunit assumes a conformation that enables the enzyme to harness the free energy of ADP binding to drive ATP release. Finally, we show that ligand exchange is regulated by a checkpoint mechanism, an apparent prerequisite for high efficiency in protein nanomotors.


Assuntos
Simulação de Dinâmica Molecular , ATPases Translocadoras de Prótons/metabolismo , Termodinâmica , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/química
14.
Nat Chem Biol ; 13(4): 363-365, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28218912

RESUMO

In this study, we engineered fatty acid synthases (FAS) for the biosynthesis of short-chain fatty acids and polyketides, guided by a combined in vitro and in silico approach. Along with exploring the synthetic capability of FAS, we aim to build a foundation for efficient protein engineering, with the specific goal of harnessing evolutionarily related megadalton-scale polyketide synthases (PKS) for the tailored production of bioactive natural compounds.


Assuntos
Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Policetídeos/metabolismo , Engenharia de Proteínas , Corynebacterium/enzimologia , Ácido Graxo Sintases/genética , Modelos Moleculares , Estrutura Molecular , Policetídeos/química
15.
J Chem Theory Comput ; 13(1): 147-160, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-27966355

RESUMO

Correct protonation of titratable groups in biomolecules is crucial for their accurate description by molecular dynamics simulations. In the context of constant pH simulations, an additional protonation degree of freedom is introduced for each titratable site, allowing the protonation state to change dynamically with changing structure or electrostatics. Here, we extend previous approaches for an accurate description of chemically coupled titrating sites. A second reaction coordinate is used to switch between two tautomeric states of an amino acid with chemically coupled titratable sites, such as aspartate (Asp), glutamate (Glu), and histidine (His). To this aim, we test a scheme involving three protonation states. To facilitate charge neutrality as required for periodic boundary conditions and Particle Mesh Ewald (PME) electrostatics, titration of each respective amino acid is coupled to a "water" molecule that is charged in the opposite direction. Additionally, a force field modification for Amber99sb is introduced and tested for the description of carboxyl group protonation. Our three states model is tested by titration simulations of Asp, Glu, and His, yielding a good agreement, reproducing the correct geometry of the groups in their different protonation forms. We further show that the ion concentration change due to the neutralizing "water" molecules does not significantly affect the protonation free energies of the titratable groups, suggesting that the three states model provides a good description of biomolecular dynamics at constant pH.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Concentração de Íons de Hidrogênio , Prótons , Solventes/química , Eletricidade Estática
16.
Nat Methods ; 14(1): 71-73, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27819658

RESUMO

The all-atom additive CHARMM36 protein force field is widely used in molecular modeling and simulations. We present its refinement, CHARMM36m (http://mackerell.umaryland.edu/charmm_ff.shtml), with improved accuracy in generating polypeptide backbone conformational ensembles for intrinsically disordered peptides and proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Dobramento de Proteína , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica
17.
J Am Chem Soc ; 138(51): 16620-16631, 2016 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-27966904

RESUMO

There has been considerable debate on the existence of a low-barrier hydrogen bond (LBHB) in the photoactive yellow protein (PYP). The debate was initially triggered by the neutron diffraction study of Yamaguchi et al. ( Proc. Natl. Acad. Sci., U. S. A. , 2009 , 106 , 440 - 444 ) who suggested a model in which a neutral Arg52 residue triggers the formation of the LBHB in PYP. Here, we present an alternative model that is consistent within the error margins of the Yamaguchi structure factors. The model explains an increased hydrogen bond length without nuclear quantum effects and for a protonated Arg52. We tested both models by calculations under crystal, solution, and vacuum conditions. Contrary to the common assumption in the field, we found that a single PYP in vacuum does not provide an accurate description of the crystal conditions but instead introduces strong artifacts, which favor a LBHB and a large 1H NMR chemical shift. Our model of the crystal environment was found to stabilize the two Arg52 hydrogen bonds and crystal water positions for the protonated Arg52 residue in free MD simulations and predicted an Arg52 pKa upshift with respect to PYP in solution. The crystal and solution environments resulted in almost identical 1H chemical shifts that agree with NMR solution data. We also calculated the effect of the Arg52 protonation state on the LBHB in 3D nuclear equilibrium density calculations. Only the charged crystal structure in vacuum supports a LBHB if Arg52 is neutral in PYP at the previously reported level of theory ( J. Am. Chem. Soc. , 2014 , 136 , 3542 - 3552 ). We attribute the anomalies in the interpretation of the neutron data to a shift of the potential minimum, which does not involve nuclear quantum effects and is transferable beyond the Yamaguchi structure.


Assuntos
Artefatos , Proteínas de Bactérias/química , Fotorreceptores Microbianos/química , Vácuo , Cristalografia por Raios X , Ligações de Hidrogênio , Modelos Moleculares , Conformação Proteica , Soluções
18.
Nature ; 540(7631): 80-85, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27842381

RESUMO

In all domains of life, selenocysteine (Sec) is delivered to the ribosome by selenocysteine-specific tRNA (tRNASec) with the help of a specialized translation factor, SelB in bacteria. Sec-tRNASec recodes a UGA stop codon next to a downstream mRNA stem-loop. Here we present the structures of six intermediates on the pathway of UGA recoding in Escherichia coli by single-particle cryo-electron microscopy. The structures explain the specificity of Sec-tRNASec binding by SelB and show large-scale rearrangements of Sec-tRNASec. Upon initial binding of SelB-Sec-tRNASec to the ribosome and codon reading, the 30S subunit adopts an open conformation with Sec-tRNASec covering the sarcin-ricin loop (SRL) on the 50S subunit. Subsequent codon recognition results in a local closure of the decoding site, which moves Sec-tRNASec away from the SRL and triggers a global closure of the 30S subunit shoulder domain. As a consequence, SelB docks on the SRL, activating the GTPase of SelB. These results reveal how codon recognition triggers GTPase activation in translational GTPases.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Códon de Terminação/química , Códon de Terminação/genética , Códon de Terminação/metabolismo , Microscopia Crioeletrônica , Endorribonucleases/metabolismo , Ativação Enzimática , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas Fúngicas/metabolismo , GTP Fosfo-Hidrolases/ultraestrutura , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , RNA de Transferência Aminoácido-Específico/química , RNA de Transferência Aminoácido-Específico/genética , RNA de Transferência Aminoácido-Específico/metabolismo , RNA de Transferência Aminoácido-Específico/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Ribossomos/química , Ribossomos/enzimologia , Ribossomos/ultraestrutura , Ricina/metabolismo , Selenocisteína/metabolismo
19.
Phys Rev Lett ; 117(18): 188102, 2016 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-27834997

RESUMO

A complete physical description of membrane remodeling processes, such as fusion or fission, requires knowledge of the underlying free energy landscapes, particularly in barrier regions involving collective shape changes, topological transitions, and high curvature, where Canham-Helfrich (CH) continuum descriptions may fail. To calculate these free energies using atomistic simulations, one must address not only the sampling problem due to high free energy barriers, but also an orthogonal sampling problem of combinatorial complexity stemming from the permutation symmetry of identical lipids. Here, we solve the combinatorial problem with a permutation reduction scheme to map a structural ensemble into a compact, nondegenerate subregion of configuration space, thereby permitting straightforward free energy calculations via umbrella sampling. We applied this approach, using a coarse-grained lipid model, to test the CH description of bending and found sharp increases in the bending modulus for curvature radii below 10 nm. These deviations suggest that an anharmonic bending term may be required for CH models to give quantitative energetics of highly curved states.


Assuntos
Bicamadas Lipídicas/química , Entropia , Membranas , Simulação de Dinâmica Molecular
20.
Biophys J ; 111(8): 1668-1678, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27760354

RESUMO

Ligand-protein binding processes are essential in biological systems. A well-studied system is the binding of cyclic adenosine monophosphate to the cyclic nucleotide binding domain of the bacterial potassium channel MloK1. Strikingly, the measured on-rate for cyclic adenosine monophosphate binding is two orders of magnitude slower than a simple Smoluchowski diffusion model would suggest. To resolve this discrepancy and to characterize the ligand-binding path in structural and energetic terms, we calculated 1100 ligand-binding molecular dynamics trajectories and tested two scenarios: In the first scenario, the ligand transiently binds to the protein surface and then diffuses along the surface into the binding site. In the second scenario, only ligands that reach the protein surface in the vicinity of the binding site proceed into the binding site. Here, a binding funnel, which increasingly confines the translational as well as the rotational degrees of freedom, determines the binding pathways and limits the on-rate. From the simulations, we identified five surface binding states and calculated the rates between these surface binding states, the binding site, and the bulk. We find that the transient binding of the ligands to the surface binding states does not affect the on-rate, such that this effect alone cannot explain the observed low on-rate. Rather, by quantifying the translational and rotational degrees of freedom and by calculating the binding committor, our simulations confirmed the existence of a binding funnel as the main bottleneck. Direct binding via the binding funnel dominates the binding kinetics, and only ∼10% of all ligands proceed via the surface into the binding site. The simulations further predict an on-rate between 15 and 40µs-1(mol/l)-1, which agrees with the measured on-rate.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , AMP Cíclico/metabolismo , Simulação de Dinâmica Molecular , Canais de Potássio/química , Canais de Potássio/metabolismo , Ligação Proteica , Domínios Proteicos , Rotação
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