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1.
Proc Natl Acad Sci U S A ; 116(12): 5356-5361, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30837309

RESUMO

As theory and experiment have shown, protein dehydration is a major contributor to protein folding. Dehydration upon folding can be characterized directly by all-atom simulations of fast pressure drops, which create desolvated pockets inside the nascent hydrophobic core. Here, we study pressure-drop refolding of three λ-repressor fragment (λ6-85) mutants computationally and experimentally. The three mutants report on tertiary structure formation via different fluorescent helix-helix contact pairs. All-atom simulations of pressure drops capture refolding and unfolding of all three mutants by a similar mechanism, thus validating the nonperturbative nature of the fluorescent contact probes. Analysis of simulated interprobe distances shows that the α-helix 1-3 pair distance displays a slower characteristic time scale than the 1-2 or 3-2 pair distance. To see whether slow packing of α-helices 1 and 3 is reflected in the rate-limiting folding step, fast pressure-drop relaxation experiments captured refolding on a millisecond time scale. These experiments reveal that refolding monitored by 1-3 contact formation indeed is much slower than when monitored by 1-2 or 3-2 contact formation. Unlike the case of the two-state folder [three-α-helix bundle (α3D)], whose drying and core formation proceed in concert, λ6-85 repeatedly dries and rewets different local tertiary contacts before finally forming a solvent-excluded core, explaining the non-two-state behavior observed during refolding in molecular dynamics simulations. This work demonstrates that proteins can explore desolvated pockets and dry globular states numerous times before reaching the native conformation.


Assuntos
Desidratação/metabolismo , Proteínas/metabolismo , Escherichia coli/metabolismo , Fluorescência , Cinética , Simulação de Dinâmica Molecular , Pressão , Conformação Proteica em alfa-Hélice/fisiologia , Dobramento de Proteína , Solventes/metabolismo
2.
Nat Nanotechnol ; 14(5): 420-425, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30833691

RESUMO

Electron microscopy has been instrumental in our understanding of complex biological systems. Although electron microscopy reveals cellular morphology with nanoscale resolution, it does not provide information on the location of different types of proteins. An electron-microscopy-based bioimaging technology capable of localizing individual proteins and resolving protein-protein interactions with respect to cellular ultrastructure would provide important insights into the molecular biology of a cell. Here, we synthesize small lanthanide-doped nanoparticles and measure the absolute photon emission rate of individual nanoparticles resulting from a given electron excitation flux (cathodoluminescence). Our results suggest that the optimization of nanoparticle composition, synthesis protocols and electron imaging conditions can lead to sub-20-nm nanolabels that would enable high signal-to-noise localization of individual biomolecules within a cellular context. In ensemble measurements, these labels exhibit narrow spectra of nine distinct colours, so the imaging of biomolecules in a multicolour electron microscopy modality may be possible.


Assuntos
Corantes Fluorescentes/química , Microscopia Eletrônica de Transmissão , Nanopartículas/química
3.
Proc Natl Acad Sci U S A ; 112(26): 7966-71, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26080403

RESUMO

Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.


Assuntos
Corantes Fluorescentes/química , Dobramento de Proteína , Cinética , Simulação de Dinâmica Molecular , Mutação , Triptofano/química , Tirosina/química
4.
J Am Chem Soc ; 137(22): 7152-7159, 2015 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-25988868

RESUMO

The unimolecular folding reaction of small proteins is now amenable to a very direct mechanistic comparison between experiment and simulation. We present such a comparison of microsecond pressure and temperature jump refolding kinetics of the engineered WW domain FiP35, a model system for ß-sheet folding. Both perturbations produce experimentally a faster and a slower kinetic phase, and the "slow" microsecond phase is activated. The fast phase shows differences between perturbation methods and is closer to the downhill limit by temperature jump, but closer to the transiently populated intermediate limit by pressure jump. These observations make more demands on simulations of the folding process than just a rough comparison of time scales. To complement experiments, we carried out several pressure jump and temperature jump all-atom molecular dynamics trajectories in explicit solvent, where FiP35 folded in five of the six simulations. We analyzed our pressure jump simulations by kinetic modeling and found that the pressure jump experiments and MD simulations are most consistent with a 4-state kinetic mechanism. Together, our experimental and computational data highlight FiP35's position at the boundary where activated intermediates and downhill folding meet, and we show that this model protein is an excellent candidate for further pressure jump molecular dynamics studies to compare experiment and modeling at the folding mechanism level.


Assuntos
Pressão , Dobramento de Proteína , Temperatura , Proteínas de Escherichia coli/química , Cinética , Simulação de Dinâmica Molecular
5.
J Am Chem Soc ; 136(50): 17547-60, 2014 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-25409346

RESUMO

PEGylation of protein side chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal losses to biological activity. We hypothesize that globally optimal PEGylation sites are characterized by the ability of the PEG oligomer to increase protein conformational stability; however, the current understanding of how PEG influences the conformational stability of proteins is incomplete. Here we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and we show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Our results highlight the possibility of using modern site-specific PEGylation techniques to install PEG oligomers at predetermined locations where PEG will provide optimal increases in conformational and proteolytic stability.


Assuntos
Polietilenoglicóis/química , Estabilidade Proteica , Proteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Dados de Sequência Molecular , Conformação Proteica , Termodinâmica
6.
J Am Chem Soc ; 136(11): 4265-72, 2014 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-24437525

RESUMO

Density is an easily adjusted variable in molecular dynamics (MD) simulations. Thus, pressure-jump (P-jump)-induced protein refolding, if it could be made fast enough, would be ideally suited for comparison with MD. Although pressure denaturation perturbs secondary structure less than temperature denaturation, protein refolding after a fast P-jump is not necessarily faster than that after a temperature jump. Recent P-jump refolding experiments on the helix bundle λ-repressor have shown evidence of a <3 µs burst phase, but also of a ~1.5 ms "slow" phase of refolding, attributed to non-native helical structure frustrating microsecond refolding. Here we show that a λ-repressor mutant is nonetheless capable of refolding in a single explicit solvent MD trajectory in about 19 µs, indicating that the burst phase observed in experiments on the same mutant could produce native protein. The simulation reveals that after about 18.5 µs of conformational sampling, the productive structural rearrangement to the native state does not occur in a single swift step but is spread out over a brief series of helix and loop rearrangements that take about 0.9 µs. Our results support the molecular time scale inferred for λ-repressor from near-downhill folding experiments, where transition-state population can be seen experimentally, and also agrees with the transition-state transit time observed in slower folding proteins by single-molecule spectroscopy.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Pressão , Redobramento de Proteína , Proteínas/genética
7.
Curr Phys Chem ; 3(2)2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24358071

RESUMO

Gold nanorods absorb and scatter light strongly in the near-infrared portion of the electromagnetic spectrum, making them ideal tissue contrast agents for imaging techniques such as optical coherence tomography (OCT). Strong interactions occur at the nano-bio interface, such as proteins binding to gold nanorods forming a 'corona.' To fulfill the promise of nanorods for applications such as contrast agents, we must better understand the intrinsic interactions of these nanomaterials with biological systems at the molecular, cellular and tissue level. In this paper, we briefly review the nanorod-protein interface. We then present some new fast relaxation imaging (FReI) measurements of how the presence of strongly-absorbing gold nanorods affects protein binding and folding, taking into account inner filter effects and the strong quenching effect of nanorods on fluorescent-labeled proteins. Next we show that two-photon photoluminescence of the gold nanorods can be used to image the nanorods in tissue constructs, allowing us to independently study their tissue distribution so they can be used successfully as contrast agents in optical coherence microscopy.

8.
Bioconjug Chem ; 24(5): 796-802, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23578107

RESUMO

Protein PEGylation is an effective method for reducing the proteolytic susceptibility, aggregation propensity, and immunogenicity of protein drugs. These pharmacokinetic challenges are fundamentally related to protein conformational stability, and become much worse for proteins that populate the unfolded state under ambient conditions. If PEGylation consistently led to increased conformational stability, its beneficial pharmacokinetic effects could be extended and enhanced. However, the impact of PEGylation on protein conformational stability is currently unpredictable. Here we show that appending a short PEG oligomer to a single Asn side chain within a reverse turn in the WW domain of the human protein Pin 1 increases WW conformational stability in a manner that depends strongly on the length of the PEG oligomer: shorter oligomers increase folding rate, whereas longer oligomers increase folding rate and reduce unfolding rate. This strong length dependence is consistent with the possibility that the PEG oligomer stabilizes the transition and folded states of WW relative to the unfolded state by interacting favorably with side-chain or backbone groups on the WW surface.


Assuntos
Peptidilprolil Isomerase/química , Polietilenoglicóis/química , Dobramento de Proteína , Humanos , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA , Conformação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína
9.
Proc Natl Acad Sci U S A ; 110(20): 8087-92, 2013 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-23620522

RESUMO

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 µs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 µs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little ß-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.


Assuntos
Bacteriófago lambda/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Proteínas Virais/química , Simulação por Computador , Temperatura Alta , Cinética , Simulação de Dinâmica Molecular , Mutação , Pressão , Espectrometria de Fluorescência , Espectrofotometria Infravermelho , Temperatura , Fatores de Tempo
10.
Biophys J ; 103(2): L20-2, 2012 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-22853917

RESUMO

Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation.


Assuntos
Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Solventes/metabolismo , Absorção , Sequência de Aminoácidos , Animais , Tampões (Química) , Dicroísmo Circular , Linguado , Transferência Ressonante de Energia de Fluorescência , Congelamento , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Água/química
11.
J Am Chem Soc ; 133(48): 19338-41, 2011 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-22066714

RESUMO

Molecular dynamics simulations combining many microsecond trajectories have recently predicted that a very fast folding protein like lambda repressor fragment λ(6-85) D14A could have a slow millisecond kinetic phase. We investigated this possibility by detecting temperature-jump relaxation to 5 ms. While λ(6-85) D14A has no significant slow phase, two even more stable mutants do. A slow phase of λ(6-85) D14A does appear in mild denaturant. The experimental data and computational modeling together suggest the following hypothesis: λ(6-85) takes only microseconds to reach its native state from an extensively unfolded state, while the latter takes milliseconds to reach compact ß-rich traps. λ(6-85) is not only thermodynamically but also kinetically protected from reaching such "intramolecular amyloids" while folding.


Assuntos
Bacteriófago lambda/química , Simulação de Dinâmica Molecular , Dobramento de Proteína , Proteínas Repressoras/química , Proteínas Virais Reguladoras e Acessórias/química , Bacteriófago lambda/genética , Mutação , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Repressoras/genética , Temperatura , Termodinâmica , Proteínas Virais Reguladoras e Acessórias/genética
12.
J Phys Chem B ; 115(9): 2090-6, 2011 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-21319829

RESUMO

Lambda repressor fragment λ(*)(6-85) is one of the fastest folding small protein fragments known to date. We hypothesized that removal of three out of five helices of λ(*)(6-85) would further reduce this protein to its smallest folding core. Molecular dynamics simulations singled out two energetically stable reduced structures consisting of only helices 1 and 4 connected by a short glycine/serine linker, as well as a less stable control. We investigated these three polypeptides and their fragments experimentally by using circular dichroism, fluorescence spectroscopy, and temperature jump relaxation spectroscopy to gain insight into their thermodynamic and kinetic properties. Based on the thermal melts, the order of peptide stability was in correspondence with theoretical predictions. The most stable two-helix bundle, λ(blue1), is a cooperatively folding miniprotein with the same melting temperature and folding rate as the full-length λ(*)(6-85) pseudo wild type and a well-defined computed structure.


Assuntos
Simulação de Dinâmica Molecular , Dobramento de Proteína , Proteínas Repressoras/química , Proteínas Virais Reguladoras e Acessórias/química , Sequência de Aminoácidos , Bases de Dados de Proteínas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Desdobramento de Proteína , Temperatura , Termodinâmica
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