A simple measure of native‐state topology and chain connectivity predicts the folding rates of two‐state proteins with and without crosslinks

Heat– and cold–denatured proteins are considered separate thermodynamic states because temperature tuning requires the protein to pass through two ‘soft’ first–order phase transitions. When both pressure and temperature changes are allowed, the heat– and cold–denatured states of proteins can be interconverted without going through the native state. This raises the question of whether these states are distinguished from one another by their folding kinetics. For the Tyr22Trp/Ala37Gly/Ala49Gly mutant of the 80 residue five–helix bundle protein λ 6−85 , we show that viscosity–corrected folding rates do not distinguish the cold– and heat–denatured states. We attribute this to a folding mechanism dominated by hydrophobic collapse. Our ‘temperature–symmetric’ approach offers an alternative to viscosity tuning with solvent additives in such cases.

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DBFOLD: An efficient algorithm for computing folding pathways of complex proteins

10.1101/2020.09.09.289116 ◽

2020 ◽

Author(s):

Amir Bitran ◽

William M. Jacobs ◽

Eugene Shakhnovich

Keyword(s):

Monte Carlo ◽

Protein Folding ◽

Detailed Balance ◽

Simulation Methods ◽

Free Energies ◽

Native State ◽

Folding Rates ◽

Computational Speed ◽

Folding Pathways

AbstractAtomistic simulations can provide valuable, experimentally-verifiable insights into protein folding mechanisms, but existing ab initio simulation methods are restricted to only the smallest proteins due to severe computational speed limits. The folding of larger proteins has been studied using native-centric potential functions, but such models omit the potentially crucial role of non-native interactions.Here, we present an algorithm, entitled DBFOLD, which can predict folding pathways for a wide range of proteins while accounting for the effects of non-native contacts. In addition, DBFOLD can predict the relative rates of different transitions within a protein’s folding pathway. To accomplish this, rather than directly simulating folding, our method combines equilibrium Monte-Carlo simulations, which deploy enhanced sampling, with unfolding simulations at high temperatures. We show that under certain conditions, trajectories from these two types of simulations can be jointly analyzed to compute unknown folding rates from detailed balance. This requires inferring free energies from the equilibrium simulations, and extrapolating transition rates from the unfolding simulations to lower, physiologically-reasonable temperatures at which the native state is marginally stable. As a proof of principle, we show that our method can accurately predict folding pathways and Monte-Carlo rates for the well-characterized Streptococcal protein G. We then show that our method significantly reduces the amount of computation time required to compute the folding pathways of large, misfolding-prone proteins that lie beyond the reach of existing direct simulation methods. Our algorithm, which is available online, can generate detailed atomistic models of protein folding mechanisms while shedding light on the role of non-native intermediates which may crucially affect organismal fitness and are frequently implicated in disease.Author summaryMany proteins must adopt a specific structure in order to function. Computational simulations have been used to shed light on the mechanisms of protein folding, but unfortunately, realistic simulations can typically only be run for small proteins, due to severe limits in computational speed. Here, we present a method to solve this problem, whereby instead of directly simulating folding from an unfolded state, we run simulations that allow for computation of equilibrium folding free energies, alongside high temperature simulations to compute unfolding rates. From these quantities, folding rates can be computed using detailed balance. Importantly, our method can account for the effects of nonnative contacts which transiently form during folding and must be broken prior to adoption of the native state. Such contacts, which are often excluded from simple models of folding, may crucially affect real protein folding pathways and are often observed in folding intermediates implicated in disease.

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Existence of DNA in yeast microbodies

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082029 ◽

1978 ◽

Vol 36 (2) ◽

pp. 232-233

Author(s):

Masako Osumi ◽

Misuzu Nagano ◽

Hiroko Kazama

Keyword(s):

Catalase Activity ◽

Buoyant Density ◽

Model System ◽

Contour Length ◽

Native State ◽

Normal Alkane ◽

Remarkable Increase ◽

Dna Molecule ◽

Mitochondrial Dnas

We have found that microbodies appeared profusely together with a remarkable increase in catalase activity in normal alkane-grown cells of hydrocarbon-utilizing Candida yeasts, and that the microbodies multiplied by division in these cells. These features of Candida yeasts seem to provide a useful model system for studies on the biogenesis of the microbody. Subsequently, we have succeeded in isolation of Candida microbodies in an apparently native state, as judged biochemically and morphologically. The presence of DNA in the purified microbody fraction thus obtained was proved by the diphenylamine method. DNA molecule of about 15 urn in contour length was released from an isolated microbody. The physicochemical analyses of the microbody DNA revealed that its buoyant density differed from nuclear and mitochondrial DNAs. All these results lead us to the possibility that there is a novel type of DNA in microbodies.

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Electron Microscopic Observation of Biological Specimens in Their Native State by Employing Cryogenic Techniques

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095315 ◽

1972 ◽

Vol 30 ◽

pp. 410-411 ◽

Cited By ~ 1

Author(s):

Tokio Nei ◽

Haruo Yotsumoto ◽

Yoichi Hasegawa ◽

Yuji Nagasawa

Keyword(s):

Electron Microscopic Observation ◽

Surface Structures ◽

Specimen Preparation ◽

Native State ◽

Electron Microscopic ◽

Biological Specimen ◽

Specimen Holder ◽

Cold Stage ◽

Preparation Techniques ◽

Scanning Electron

In order to observe biological specimens in their native state, that is, still containing their water content, various methods of specimen preparation have been used, the principal two of which are the chamber method and the freeze method.Using its recently developed cold stage for installation in the pre-evacuation chamber of a scanning electron microscope, we have succeeded in directly observing a biological specimen in its frozen state without the need for such conventional specimen preparation techniques as drying and metallic vacuum evaporation. (Echlin, too, has reported on the observation of surface structures using the same freeze method.)In the experiment referred to herein, a small sliced specimen was place in the specimen holder. After it was rapidly frozen by freon cooled with liquid nitrogen, it was inserted into the cold stage of the specimen chamber.

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