Ultrafast Proteinquake Dynamics in Cytochrome<i>c</i>

Zang, Chen; Stevens, Jeffrey A.; Link, Justin; Guo, Lijun; Wang, Limin; Zhong, Dongping

doi:10.1021/ja8057293

Cited by 53 publications

(64 citation statements)

References 71 publications

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“…When the local region around Met80 unfolds, this absorption band is absent because of the breakage of the Met80 to heme bond. The identification of the Ω–loop containing Met80 as being a least stable region, or weakest link in the protein, came from earlier residue-resolved hydrogen exchange (HX) experiments measured using two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy 28–31 , protein unfolding monitored in response to various stresses 32–34 , and from studies on ultrafast protein dynamics 35 . Our earlier computational analysis predicts that the six residues around Met80 acts as an aggregation “hot-spot” whose unfolding may lead to Cyt c aggregation 36 , and experimental results confirm this hypothesis 36 .…”

Section: Introductionmentioning

confidence: 99%

Effect of Antimicrobial Preservatives on Partial Protein Unfolding and Aggregation

Hutchings

Singh

Cabello-Villegas

et al. 2013

Journal of Pharmaceutical Sciences

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One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation “hot-spot” reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation “hot-spot”. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the “hot-spot” reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the “hot-spot” for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others.

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Section: Introductionmentioning

confidence: 99%

Effect of Antimicrobial Preservatives on Partial Protein Unfolding and Aggregation

Hutchings

Singh

Cabello-Villegas

et al. 2013

Journal of Pharmaceutical Sciences

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“…A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2)(3)(4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5)(6)(7)(8)(9)(10)(11).…”

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confidence: 99%

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Haque

Stuehr

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Mechanisms that regulate the nitric oxide synthase enzymes (NOS) are of interest in biology and medicine. Although NOS catalysis relies on domain motions, and is activated by calmodulin binding, the relationships are unclear. We used single-molecule fluorescence resonance energy transfer (FRET) spectroscopy to elucidate the conformational states distribution and associated conformational fluctuation dynamics of the two electron transfer domains in a FRET dye-labeled neuronal NOS reductase domain, and to understand how calmodulin affects the dynamics to regulate catalysis. We found that calmodulin alters NOS conformational behaviors in several ways: It changes the distance distribution between the NOS domains, shortens the lifetimes of the individual conformational states, and instills conformational discipline by greatly narrowing the distributions of the conformational states and fluctuation rates. This information was specifically obtainable only by single-molecule spectroscopic measurements, and reveals how calmodulin promotes catalysis by shaping the physical and temporal conformational behaviors of NOS.A lthough proteins adopt structures determined by their amino acid sequences, they are not static objects and fluctuate among ensembles of conformations (1). Transitions between these states can occur on a variety of length scales (Å to nm) and time scales (ps to s) and have been linked to functionally relevant phenomena such as allosteric signaling, enzyme catalysis, and protein-protein interactions (2-4). Indeed, protein conformational fluctuations and dynamics, often associated with static and dynamic inhomogeneity, are thought to play a crucial role in biomolecular functions (5-11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solution by an ensemble-averaged measurement, especially in proteins that undergo multiple-conformation transformations. In such cases, single-molecule spectroscopy is a powerful approach to analyze protein conformational states and dynamics under physiological conditions, and can provide a molecular-level perspective on how a protein's structural dynamics link to its functional mechanisms (12-21).A case in point is the nitric oxide synthase (NOS) enzymes (22-24), whose nitric oxide (NO) biosynthesis involves electron transfer reactions that are associated with relatively large-scale movement (tens of Å) of the enzyme domains (Fig. 1A). During catalysis, NADPH-derived electrons first transfer into an FAD domain and an FMN domain in NOS that together comprise the NOS reductase domain (NOSr), and then transfer from the FMN domain to a heme group that is bound in a separate attached "oxygenase" domain, which then enables NO synthesis to begin (22,(25)(26)(27). The electron transfers into and out of the FMN domain are the key steps for catalysis, and they appear to rely on the FMN domain cycling between electron-accepting and electron-donating conformational states (28, 29) (Fig. 1B). In this model, the FMN domain is suggested to be highly...

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“…[5][6][7] Typically, relaxation times are studied via an ultrafast ͑femtosecond or picosecond͒ pump-probe technique by measuring either fluorescence in a reflection configuration or absorption in a transmission configuration. 8,9 In the reflection-based approach, the molecules are excited by a laser pulse through the transition ͉0͘ → ͉i͘.…”

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confidence: 99%

Picosecond absorption relaxation measured with nanosecond laser photoacoustics

Danielli

Favazza

Maslov

et al. 2010

Applied Physics Letters

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Picosecond absorption relaxation-central to many disciplines-is typically measured by ultrafast ͑femtosecond or picosecond͒ pump-probe techniques, which however are restricted to optically thin and weakly scattering materials or require artificial sample preparation. Here, we developed a reflection-mode relaxation photoacoustic microscope based on a nanosecond laser and measured picosecond absorption relaxation times. The relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, were measured at 576 nm. The added advantages in dispersion susceptibility, laser-wavelength availability, reflection sensing, and expense foster the study of natural-including strongly scattering and nonfluorescent-materials. © 2010 American Institute of Physics. ͓doi:10.1063/1.3500820͔Picosecond absorption relaxation is central to many optical phenomena in physics, chemistry, biology, and medicine-such as absorption saturation, 1-3 photosynthesis, 4 and photolysis. [5][6][7] Typically, relaxation times are studied via an ultrafast ͑femtosecond or picosecond͒ pump-probe technique by measuring either fluorescence in a reflection configuration or absorption in a transmission configuration. 8,9 In the reflection-based approach, the molecules are excited by a laser pulse through the transition ͉0͘ → ͉i͘. A probe pulse with a variable delay time relative to the pump pulse monitors the time evolution of the population density N i ͑t͒. 8 When applied to molecules with low fluorescence quantum yield, this approach is noisy. In the transmission-based approach, the relaxation time is extracted by measuring pump-induced changes in probe-beam transmittivity ⌬T = T͑I p ͒ − T͑0͒, where T͑I p ͒ and T͑0͒ are the probe beam transmittivities with and without a pump of intensity I p . 9 In both approaches, the time resolution is limited by the pulse width of both the pump and the probe pulses. Hence, not only is an expensive picosecond or femtosecond laser required, but also the detection is susceptible to pulse broadening in dispersive media. Alternatively, intensity correlation of nanosecond laser pulses can be used. 10,11 Working in transmission mode, this technique is not suitable for optically thick media. All of the current techniques are limited to optically thin and weakly scattering materials or require artificial sample preparation.Photoacoustic microscopy and photoacoustic computed tomography, usually based on nanosecond laser excitation, are effective functional and molecular imaging tools in vivo. In the photoacoustic phenomenon, light is absorbed by a material and converted to heat. The subsequent thermoelastic expansion generates a detectable acoustic wave. 12 Photoacoustic sensing presents an exquisite sensitivity due to its inherent background-free nature. Its relative sensitivity to absorption reaches the theoretical limit of 100%, by far the highest among all optical imaging modalities. Most quantitative photoacoustic studies have assumed a linear dependence between ...

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Ultrafast Proteinquake Dynamics in Cytochromec

Cited by 53 publications

References 71 publications

Effect of Antimicrobial Preservatives on Partial Protein Unfolding and Aggregation

Effect of Antimicrobial Preservatives on Partial Protein Unfolding and Aggregation

Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics

Picosecond absorption relaxation measured with nanosecond laser photoacoustics

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