scholarly journals Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin

2016 ◽  
Author(s):  
Govardhan Reddy ◽  
D. Thirumalai
Keyword(s):  
Single Molecule ◽  
Quantitative Agreement ◽  
Coarse Grained ◽  
Radius Of Gyration ◽  
Structural Elements ◽  
Neutral Ph ◽  
X Ray Scattering ◽  
Unfolded State ◽  
Polypeptide Chains ◽  
Kinetics Of

AbstractDespite the small size the folding of Ubiquitin (Ub), which plays an indispensable role in targeting proteins for degradation and DNA damage response, is complex. A number of experiments on Ub folding have reached differing conclusions regarding the relation between collapse and folding, and whether intermediates are populated. In order to resolve these vexing issues, we elucidate the denaturant-dependent thermodynamics and kinetics of Ub folding in low and neutral pH as a function of Guanidinium chloride and Urea using coarse-grained molecular simulations. The changes in the fraction of the folded Ub, and the radius of gyration (Rg) as a function of the denaturant concentration, [C], are in quantitative agreement with experiments. Under conditions used in experiments,Rgof the unfolded state at neutral pH changes only by ≈ 17% as the [GdmCl] decreases from 6 M to 0 M. We predict that the extent of compaction of the unfolded state increases as temperature decreases. A two-dimensional folding landscape as a function ofRgand a measure of similarity to the folded state reveals unambiguously that the native state assembly is preceded by collapse, as discovered in fast mixing experiments on several proteins. Analyses of the folding trajectories, under mildly denaturing conditions ([GdmCl]=1.0M or [Urea]=1.0M), shows that Ub folds by collision between preformed secondary structural elements involving kinetic intermediates that are primarily stabilized by long-range contacts. Our work explains the results of Small Angle X-Ray Scattering (SAXS) experiments on Ub quantitatively, and establishes that evolved globular proteins are poised to collapse. In the process, we explain the discrepancy between SAXS and single molecule fluorescent resonant energy transfer (smFRET) experiments, which have arrived at a contradicting conclusion concerning the collapse of polypeptide chains.

scholarly journals Folding PDZ2 domain using the Molecular Transfer Model

2016 ◽  
Author(s):  
Zhenxing Liu ◽  
Govardhan Reddy ◽  
Dave Thirumalai
Keyword(s):  
Pdz Domain ◽  
High Energy ◽  
Coarse Grained ◽  
Radius Of Gyration ◽  
Transfer Model ◽  
Self Organized ◽  
Energy Profiles ◽  
X Ray Scattering ◽  
Molecular Transfer

A major challenge in molecular simulations is to describe denaturant-dependent folding of proteins order to make direct comparisons within vitroexperiments. We use the molecular transfer model (MTM), which is currently the only method that accomplishes this goal albeit phenomenologically, to quantitatively describe urea-dependent folding of PDZ domain, which plays a significant role in molecular recognition and signaling. Experiments show that urea-dependent unfolding rates of the PDZ2 domain exhibit a downward curvature at high urea concentrations ([C]s), which has been interpreted by invoking the presence of a sparsely populated high energy intermediate. Simulations using the MTM and a coarse-grained Self-Organized Polymer (SOP) representation of PDZ2 are used to show that the intermediate (IEQ), which has some native-like character, is present in equilibrium both in the presence and absence of urea. The free energy profiles as a function of the structural overlap order parameter show that there are two barriers separating the folded and unfolded states. Structures of the transition state ensembles, (TSE1 separating the unfolded and (IEQ) andTSE2 separatingIEQand the native state), determined using thePfoldmethod, show thatTSE1 is greatly expanded whileTSE2 is compact and native-like. Folding trajectories reveal that PDZ2 folds by parallel routes. In one pathway folding occurs exclusively throughI1, which resemblesIEQ. In a fraction of trajectories, constituting the second pathway, folding occurs through a combination ofI1and a kinetic intermediate. We establish that the radius of gyration (RUg) of the unfolded state is more compact (by ∼9%) under native conditions. Theory and simulations show that the decrease inRUgoccurs on the time scale on the order of utmost ∼20μs. The modest decrease inRUgand the rapid collapse suggest that high spatial and temporal resolution, currently beyond the scope of most small angle X-ray scattering experiments, are needed to detect compaction in finite-sized proteins. The present work further establishes that MTM is efficacious in producing nearly quantitative predictions for folding of proteins under conditions used to carry out experiments.

Pore growth kinetics in heat-treated glass-like carbons

1986 ◽  
Vol 1 (6) ◽  
pp. 832-835 ◽  
Author(s):  
Jalil Lachter ◽  
Leo G. Henry ◽  
Robert H. Bragg ◽  
Stephen Spooner
Keyword(s):  
Growth Kinetics ◽  
Matrix Material ◽  
Radius Of Gyration ◽  
Void Size ◽  
Kcal Mole ◽  
X Ray ◽  
X Ray Scattering ◽  
Heat Treated ◽  
The Matrix ◽  
Kinetics Of

The kinetics of changes in void size during annealing of glass-like carbons in the temperature range 1000°−2800 °C for times up to 150 h were studied using small-angle x-ray scattering determinations of the radius of gyration Rg. The results show that Rg ranged from 9 Å at 1000°C to about 24 Å at 2800 °C. A pore coarsening analysis and a superimposition kinetic analysis applied to Rg gave activation energies of 76 ± 4 kcal/mole and 74 ± 9 kcal/mole, respectively, which are associated with migration of vacancies within graphitic layers in the matrix material.

scholarly journals Dissecting the self-assembly kinetics of multimeric pore-forming toxins

2016 ◽  
Vol 13 (114) ◽  
pp. 20150762 ◽  
Author(s):  
A. A. Lee ◽  
M. J. Senior ◽  
M. I. Wallace ◽  
T. E. Woolley ◽  
I. M. Griffiths
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Quantitative Agreement ◽  
Time Resolved ◽  
Biologically Relevant ◽  
Low Concentrations ◽  
Assembly Kinetics ◽  
Quantitative Understanding ◽  
Mechanism And Kinetics ◽  
Kinetics Of

Pore-forming toxins are ubiquitous cytotoxins that are exploited by both bacteria and the immune response of eukaryotes. These toxins kill cells by assembling large multimeric pores on the cell membrane. However, a quantitative understanding of the mechanism and kinetics of this self-assembly process is lacking. We propose an analytically solvable kinetic model for stepwise, reversible oligomerization. In biologically relevant limits, we obtain simple algebraic expressions for the rate of pore formation, as well as for the concentration of pores as a function of time. Quantitative agreement is obtained between our model and time-resolved kinetic experiments of Bacillus thuringiensis Cry1Ac (tetrameric pore), aerolysin, Staphylococcus aureus α -haemolysin (heptameric pores) and Escherichia coli cytolysin A (dodecameric pore). Furthermore, our model explains how rapid self-assembly can take place with low concentrations of oligomeric intermediates, as observed in recent single-molecule fluorescence experiments of α-haemolysin self-assembly. We propose that suppressing the concentration of oligomeric intermediates may be the key to reliable, error-free, self-assembly of pores.

scholarly journals Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

2019 ◽  
Vol 116 (25) ◽  
pp. 12301-12310 ◽  
Author(s):  
Ivan Peran ◽  
Alex S. Holehouse ◽  
Isaac S. Carrico ◽  
Rohit V. Pappu ◽  
Osman Bilsel ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Random Coil ◽  
Coarse Grained ◽  
Unfolded Proteins ◽  
Time Resolved ◽  
Conformational Preferences ◽  
Unfolded State ◽  
Theoretical Predictions

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

scholarly journals Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Salvatore Assenza ◽  
Alberto Stefano Sassi ◽  
Ruth Kellner ◽  
Benjamin Schuler ◽  
Paolo De Los Rios ◽  
...  
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Quantitative Agreement ◽  
Mechanical Work ◽  
Chemical Energy ◽  
Coarse Grained ◽  
Theoretical Approaches ◽  
Non Equilibrium

Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here, we combine complementary theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to effectively convert chemical energy into mechanical work close to physiological conditions.

scholarly journals On the Importance of Hydrodynamic Interactions in the Stepping Kinetics of Kinesin

2016 ◽  
Author(s):  
Dave Thirumalai ◽  
Yonathan Goldtzvik ◽  
Zhechun Zhang
Keyword(s):  
Coiled Coil ◽  
Quantitative Agreement ◽  
Single Step ◽  
Hydrodynamic Interactions ◽  
Coarse Grained ◽  
Coarse Grained Model ◽  
Target Binding ◽  
Conventional Kinesin ◽  
Kinetics Of ◽  
Target Binding Site

Conventional kinesin walks by a hand-over-hand mechanism on the microtubule (MT) by taking ∼ 8nmdiscrete steps, and consumes one ATP molecule per step. The time needed to complete a single step is on the order of twenty microseconds. We show, using simulations of a coarse-grained model of the complex containing the two motor heads, the MT, and the coiled coil that in order to obtain quantitative agreement with experiments for the stepping kinetics hydrodynamic interactions (HI) have to be included. In simulations without hydrodynamic interactions spanning nearly twenty microseconds not a single step was completed in hundred trajectories. In sharp contrast, nearly 14% of the steps reached the target binding site within 6 microseconds when HI were included. Somewhat surprisingly, there are qualitative differences in the diffusion pathways in simulations with and without HI. The extent of movement of the trailing head of kinesin on the MT during the diffusion stage of stepping is considerably greater in simulations with HI than in those without HI. Our results suggest that inclusion of HI is crucial in the accurate description of motility of other motors as well.

scholarly journals Small-Angle X-ray Scattering and Single-Molecule FRET Spectroscopy Produce Highly Divergent Views of the Low-Denaturant Unfolded State

2012 ◽  
Vol 418 (3-4) ◽  
pp. 226-236 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Steve P. Meisburger ◽  
James Hinshaw ◽  
Lois Pollack ◽  
Gilad Haran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Small Angle ◽  
X Ray ◽  
Single Molecule Fret ◽  
X Ray Scattering ◽  
Unfolded State ◽  
Ray Scattering

m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation Complement and Fibrinolytic Serine Proteases

1979 ◽  
Author(s):  
D Bing ◽  
D Robison ◽  
J Andrews ◽  
R Laura
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inhibitor ◽  
Molecular Model ◽  
Factor Xa ◽  
Affinity Labeling ◽  
Catalytic Center ◽  
Inhibitor Complex ◽  
Polypeptide Chains ◽  
Kinetics Of

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F] is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component CIS and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 x 10-3sec-1), the kinetics of inactivation are consistent with inhibition proceding via formation of a specific enzyme-inhibitor complex analogous to a Michaelis-Menton complex (KL - 115-26 μM), and diisopropylfluorophosphate or p-amidino-phenylmethanesulfonyfluoride Prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cationic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants

In situ Time-Resolved Small-Angle X-ray Scattering Reveals the Formation Kinetics of Polyelectrolyte Complex Micelles

2019 ◽  
Author(s):  
Hao Wu ◽  
Jeffrey Ting ◽  
Siqi Meng ◽  
Matthew Tirrell
Keyword(s):  
Small Angle ◽  
Self Assembly ◽  
Electrostatic Interactions ◽  
Polyelectrolyte Complex ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Kinetics Of ◽  
Ray Scattering

We have directly observed the <i>in situ</i> self-assembly kinetics of polyelectrolyte complex (PEC) micelles by synchrotron time-resolved small-angle X-ray scattering, equipped with a stopped-flow device that provides millisecond temporal resolution. This work has elucidated one general kinetic pathway for the process of PEC micelle formation, which provides useful physical insights for increasing our fundamental understanding of complexation and self-assembly dynamics driven by electrostatic interactions that occur on ultrafast timescales.

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