Alan fersht structure and mechanism in protein science pdf file

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alan fersht structure and mechanism in protein science pdf file

File Name: alan fersht structure and mechanism in protein science

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Published: 12.05.2021

Fersht was born on 21 April [23] in Hackney , London.

Protein Folding Kinetics

The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such un folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the un folding reaction has often been questioned. One of the most well studied un folding pathways is that of Drosophila melanogaster Engrailed homeodomain EnHD : this residue protein forms a three helix bundle in the native state and folds via a helical intermediate.

The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed un folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the un folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions.

Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution. The folding of proteins to their functional conformations has been studied extensively both experimentally and through theoretical simulations. Nevertheless, there are several cases where intermediates accumulate in protein un folding reactions as on-pathway species [ 4 — 8 ].

There is however an ongoing debate about whether these intermediates are productive in the strict definition that they are obligatory species on the path to the native state. Among fast-folding proteins, Drosophila melanogaster Engrailed homeodomain EnHD is one of the best studied systems.

A combination of experimental and computational methods have demonstrated that EnHD folds by initial formation of secondary structure elements that subsequently dock to form the native state [ 9 , 10 ] in line with the diffusion-collision model [ 11 ]. Protein engineering in combination with nuclear magnetic resonance NMR showed that this un folding intermediate or denatured state under physiological conditions, D phys contains both native and non-native helices [ 12 ], and that the helix-turn-helix motif constituting H2-H3 forms independently of H1 [ 13 ], and can thus be regarded the main structural unit of the intermediate.

It is in general not feasible to directly extract 3D atomic structures from disordered systems. Structural intermediates have uniquely been identified for smaller molecules in several time-resolved WAXS experiments [ 16 — 18 ] demonstrating the achievable resolution of the technique. WAXS has recently been extended to also probe the rearrangement of secondary structural elements within proteins [ 19 ]. In addition, a methodology based on refining the molecule of interest toward solution scattering data using MD simulation has very recently been developed and successfully applied on several molecular systems [ 20 ].

H N -N residual dipolar couplings RDCs are highly sensitive to the orientations of amide bond vectors within the molecular frame [ 21 , 22 ].

Small changes in the relative bond vector orientation within that frame will result in a coupling different from that predicted from the reference structure. A poor correlation between measured and predicted data suggests a change in secondary or tertiary structures. These ensembles correspond to the denatured state D, an un folding intermediate I, corresponding to the denatured state under physiological conditions and the native state N.

The EnHD protein was expressed and purified as previously described [ 30 ]. A solution of 1. Part of the tubing and the capillary was placed in-between two metal plates with a small hole for the X-ray to enter the capillary. The temperature was regulated by a thermo coupler and a cooling system integrated in the metal plates. About 30 cm of tubing on each side of the capillary was inserted in between the plates to ensure that the whole sample volume was heated to the desired temperature.

During the acquisition cycle a sequence of scattering images were recorded using an integration time of 7 ms and a readout delay of 3 ms, equating to a readout frequency of Hz. Pixel masking and radial integration of each Pilatus frame was computed at the beamline by in house software. A MATLAB script specifically written for this experiment was later utilized to filter out outliers mainly caused by bubbles or aggregates in the solution , to normalize the intensity of the single azimuthally integrated frame to the incoming flux, for the statistical analysis, and to subtract the buffer signal from the protein solution.

Traditional Guinier plots, at q spanning from 0. At q -values below 0. The H N -N residual dipolar couplings were measured as described previously [ 12 ] in 20 mM sodium acetate, pH 5.

The conditions for the measurements are slightly different from those in the SAXS measurements in order to lower the stability of EnHD and ensure that the RDCs can be measured past the Tm of the protein on our Bruker DRX spectrometer equipped with a single axis gradient cryo-probe. The protein molecule was immersed in a periodic box containing water molecules. A cutoff distance 1 nm has been used for calculations of Lennard-Jones interactions and PME was used for the treatment of the long-range electrostatic interactions.

An integration time step of 2 fs was used and the Berendsen algorithm for temperature and pressure control [ 37 ] was used with 0. The pressure was kept at 1 atm. In total eight trajectories of each ns were generated. The temperature for each production run was , , , , , , and K. Before the production run, the system was minimized using steepest descent for steps after which the system was equilibrated in dynamic simulation 20 ps long with 1 fs time step at K.

Atomic coordinates from the last equilibration snapshot were then used as an input for the production runs at different temperature. Protein structures for fitting to the experimental data were obtained after cluster analysis on each MD trajectory. Clustering has been accomplished by the algorithm due to Daura et al. Structures for the cluster analysis were sampled from MD-trajectory every 10 ps, for a total of structures from each trajectory. As the clustering criterion, the root mean square deviation RMSD of main-chain and C-beta atoms was used.

Cut-off clustering distances of 1. Only clusters consisting of ten or more structures have been taken into account for SAXS fitting. The representative structure for each cluster was taken to be the structure from the MD-trajectory most closely located to the center of the cluster in RMSD space. Theoretical spectra of X-ray scattering and violations from NMR restraints were calculated from the central cluster structures.

X-ray scattering was computed using the CRYSOL [ 39 ] software in the range of reciprocal space corresponding to the experimental data. Due to the small size of the protein and fast sidechain dynamics, the solvent shell contrast around the protein has been kept to zero [ 14 ].

The maximum order of harmonics, which defines the resolution of the scattering curve, was set to seven. The power n is usually taken between three and six, where the higher number gives better correlation between calculated and experimental distance restrains [ 41 ]. The ensemble optimization approach by Bernado and co-workers [ 42 ] was utilized to obtain the ensemble of protein structures that reproduced the experimental scattering data using an iterative genetic algorithm [ 43 ].

Ensembles were formed from an extensive pool of conformers generated by MD simulations. The fitting was performed to reproduce the logarithm of the experimental scattering intensity, log I q. The square difference between experimental and ensemble-averaged X-ray scattering was used as the target function in the fitting. At each step of the algorithm several ensembles of structures, denominated chromosomes, were tested and sorted according to the value of the target function.

The specified number of ensembles showing the lowest values of target function was selected to pass into the next iteration. The size of each ensemble had a maximum of 20 spectra. All spectra were of equal weight in the chromosome, yet multiple repetitions of structures were allowed, which gives the possibility to change the partial weights of the structures that contributes to the ensemble. Optimization was performed for iterations and repeated 50 times. In total 20 ensembles were selected to pass through each iteration of the algorithm.

The crossover operator was tuned to generate the same number of chromosomes as the initial population. The number of chromosomes generated by the mutation operator exceeded the number of initial chromosomes by the factor of two. This last option was selected in order to achieve rapid convergence toward the optimal solution due to higher ensemble divergence created by the mutation operator.

The code for the optimization algorithm was implemented in a MATLAB package and is available from the authors upon request. MD simulations are a powerful tool to generate physical conformations of biomolecules.

The part of phase space that is sampled depends predominantly on the temperature applied. The conformations sampled in simulations of EnHD at 8 different temperatures were projected on a plane with the radius of gyration on the X-axis and the root mean square deviation from the NMR structure on the Y-axis Fig 1.

Thus, the simulations show a large diversity of compact structures in the molecular species resulting from thermal un folding. The highest temperatures yield the most unfolded structures within the relatively short ns simulation time. Since the structures are generated just for sampling phase space, the fact that the temperatures of the simulation are much higher than those used in the experiments is irrelevant.

The plot also shows the correlation between radius of gyration and root mean square deviation from the NMR structure. Conventional SAXS experiments give information mainly about the overall shape and size of proteins in solution. By extending the collected scattering angles, into the range between 0. In an optimization procedure [ 42 ], based on an iterative genetic algorithm [ 43 ], the ensemble of protein structures that best reproduced the experimental data was selected from a pool of MD and NMR structures as is further discussed in Materials and Methods.

X-ray scattering 0. The error bar at e indicates a standard deviation obtained in ensemble fitting. Scattering profiles at a and d and weight distribution at b are shifted to increase visibility. The scattering profiles Fig 2a show a pronounced broad peak at around 0. This pattern provides the signature of a significant shift in the protein population towards less folded structures at the melting point. Fig 2b shows the radius of gyration of the protein structure ensemble obtained from the fitting to the scattering data using the optimization algorithm see Materials and Methods.

Three groups of structures can be identified. The second group yellow contains proteins with less organized structures with radii of gyration between The third group red at higher radii of gyration contains denatured structures where some of the helices are completely unfolded.

In Fig 2c a structural comparison of the four most abundant structures in each group is displayed. The numbering within each group is related to the relative weight of these structures in the fitting at most temperatures. Fig 2d shows the calculated scattering profiles for the most abundant structures in each group. A clear difference in scattering signatures between the three groups of structures is apparent, and it is obvious that the structure seen around 0.

In Fig 2e the relative weight of the three groups at the four temperatures is shown. As expected, a transition from native like structures towards more unfolded structures with increasing temperature is observed. For a fully unfolded protein, the H N -N RDCs in radially compressed acrylamide gels will be slightly negative, and there will be no agreement between the measured RDCs and the crystal structure across all of the protein [ 44 ].

This gives new insights into the structural complexity of protein un folding in Engrailed Homeodomain. From the fitting of the X-ray scattering data we can resolve three groups of structures that seem to be present at all temperatures but with varying occupancy.

Few structures are presented in the optimized ensembles, which is not surprising due to high stability of the protein and fast convergence of the optimization algorithm. The other three structures in the native group are quite similar to the NMR structure where the main differences are located at the termini. In the intermediate group the helices are usually maintained with a few exceptions such as I2 for which two of the helices, H1 and H2, are more or less unfolded.

Similar number of violated restraints was obtained for NMR-refined and crystal structures [ 12 ].

Testing protein-folding simulations by experiment: B domain of protein A.

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Protein Structure Pdf

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