Mutation in a non-force-bearing region of protein L influences force-dependent unfolding behavior

Single-molecule magnetic tweezers(MTs) have revealed multiple transition barriers along the unfolding pathway of several two-state proteins, such as GB1 and Csp. In this study, we utilized MTs to measure the force-dependent folding and unfolding rates of both protein L(PLWT) and its Y47W mutant(PLY47W) where the mutation point is not at the force-bearing β-strands. The measurements were conducted within a force range of 3–120 pN. Notably, the unfolding rates of both PLWT and PWY47W exhibit distinct force sensitivities below 50 pN and above 60 pN, implying a two-barrier free energy landscape. Both PLWT and PLY47W share the same force-dependent folding rate and the same transition barriers,but the unfolding rate of PLY47W is faster than that of PLWT. Our finding demonstrates that the residue outside of the force-bearing region will also affect the force-induced unfolding dynamics.

Effect of chaperone–client interaction strength on Hsp70-mediated protein folding

Protein folding in crowding cellular environment often relies on the assistance of various chaperones. Hsp70 is one of the most ubiquitous chaperones in cells. Previous studies showed that the chaperone–client interactions at the open state tend to remodel the protein folding energy landscape and direct the protein folding as a foldase. In this work, we further investigate how the chaperone–client interaction strength modulates the foldase function of Hsp70 by using molecular simulations. The results showed that the time of substrate folding(including the whole folding step and substrate release step) has a non-monotonic dependence on the interaction strength. With the increasing of the chaperone–client interaction strength, the folding time decreases first, and then increases. More detailed analysis showed that when the chaperone–client interaction is too strong, even small number of chaperones–client contacts can maintain the substrate bound with the chaperone. The sampling of the transient chaperones–client complex with sparse inter-molecule contacts makes the client protein have chance to access the misfolded state even it is bound with chaperone. The current results suggest that the interaction strength is an important factor controlling the Hsp70 chaperoning function.

Does mRNA structure contain genetic information for regulating co-translational protein folding？

Currently many facets of genetic information are illdefined. In particular, how protein folding is genetically regulated has been a long-standing issue for genetics and protein biology. And a generic mechanistic model with supports of genomic data is still lacking. Recent technological advances have enabled much needed genome-wide experiments. While putting the effect of codon optimality on debate, these studies have supplied mounting evidence suggesting a role of m RNA structure in the regulation of protein folding by modulating translational elongation rate. In conjunctions with previous theories, this mechanistic model of protein folding guided by m RNA structure shall expand our understandings of genetic information and offer new insights into various biomedical puzzles.

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

Prediction of protein folding rates from primary sequence by fusing multiple sequential features

We have developed a web-server for predicting the folding rate of a protein based on its amino acid sequence information alone. The web- server is called Pred-PFR (Predicting Protein Folding Rate). Pred-PFR is featured by fusing multiple individual predictors, each of which is established based on one special feature derived from the protein sequence. The ensemble pre-dictor thus formed is superior to the individual ones, as demonstrated by achieving higher correlation coefficient and lower root mean square deviation between the predicted and observed results when examined by the jack-knife cross-validation on a benchmark dataset constructed recently. As a user-friendly web- server, Pred-PFR is freely accessible to the public at www.csbio.sjtu.edu.cn/bioinf/Folding Rate/.

Respective Roles of Short-and Long-Range Interactions in Protein Folding

A new method was presented to discuss the respective roles of short- and long-range interactions in protein folding. It's based on an off-lattice model, which is also being called as toy model. Simulated annealing algorithm was used to search its native conformation. When it is applied to analysis proteins 1agt and 1aho, we find that helical segment cannot fold into native conformation without the influence of long-range interactions. That's to say that long-range interactions are the main determinants in protein folding. Key words toy model - protein folding - simulated annealing algorithm - short and long range interactions CLC number O 242.28 - Q71 Foundation item: Supported by the National Natural Science Foundation of China((60301009)Biography: WANG Long-hui (1976-), female, Ph. D candidate, research direction: machine learning, bioinformatics.

Mixed Search Algorithm for Protein Folding

Computer simulations of simple exact lattice models are an aid in the study of protein folding process. We proposed a new search strategy by two hierarchy optimization techniques, which is shown very efficient.

Heuristic algorithm for off-lattice protein folding problem

Enlightened by the law of interactions among objects in the physical world, we propose a heuristic algorithm for solving the three-dimensional (3D) off-lattice protein folding problem. Based on a physical model, the problem is converted from a nonlinear constraint-satisfied problem to an unconstrained optimization problem which can be solved by the well-known gra- dient method. To improve the efficiency of our algorithm, a strategy was introduced to generate initial configuration. Computa- tional results showed that this algorithm could find states with lower energy than previously proposed ground states obtained by nPERM algorithm for all chains with length ranging from 13 to 55.

Folding rate prediction using complex network analysis for proteins with two- and three-state folding kinetics

It is a challenging task to investigate the different in- fluence of long-range and short-range interactions on two-state and three-state folding kinetics of protein. The networks of the 30 two-state proteins and 15 three-state proteins were constructed by complex networks analysis at three length scales: Protein Contact Networks, Long-range Interaction Networks and Short-range Interaction Networks. To uncover the relationship between structural properties and folding kinetics of the proteins, the correlations of protein network parameters with protein folding rate and topology parameters contact order were analyzed. The results show that Protein Contact Networks and Short-range Interaction Networks (for both two-state and three-state proteins) exhibit the “small-world” property and Long-range Interaction networks indicate “scale-free” behavior. Our results further indicate that all Protein Contact Networks and Short- range Interaction networks are assortative type. While some of Long-range Interaction Networks are of assortative type, the others are of disassortative type. For two-state proteins, the clustering coefficients of Short-range Interaction Networks show prominent correlation with folding rate and contact order. The assortativity coefficients of Short-range Interaction Networks also show remarkable correlation with folding rate and contact order. Similar correlations exist in Protein Contact Networks of three-state proteins. For two-state proteins, the correlation between contact order and folding rate is determined by the numbers of local contacts. Short- range interactions play a key role in determining the connecting trend among amino acids and they impact the folding rate of two-state proteins directly. For three-state proteins, the folding rate is determined by short-range and long-range interactions among residues together.

A Simulated Annealing Algorithm for Training Empirical Potential Functions of Protein Folding

In this paper are reported the local minimum problem by means of current greedy algorithm for training the empirical potential function of protein folding on 8623 non-native structures of 31 globular proteins and a solution of the problem based upon the simulated annealing algorithm. This simulated annealing algorithm is indispensable for developing and testing highly refined empirical potential functions.

Equilibrium folding and unfolding dynamics to reveal detailed free energy landscape of src SH3 protein by magnetic tweezers

Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics.Force spectroscopy experiments by optical tweezers and atomic force microscopy have measured the force-dependent unfolding rates with different kinds of pulling geometry.However,the equilibrium folding and unfolding dynamics at constant forces has not been reported.Here,using stable magnetic tweezers,we performed equilibrium folding and unfolding dynamic measurement and force-jump measurement of src SH3 domain with tethering points at its N-and C-termini.From the obtained force-dependent transition rates,a detailed two-state free energy landscape of src SH3 protein is constructed with quantitative information of folding free energy,transition state barrier height and position,which exemplifies the capability of magnetic tweezers to study protein folding and unfolding dynamics.

Application of topological soliton in modeling protein folding: Recent progress and perspective

Proteins are important biological molecules whose structures are closely related to their specific functions. Understanding how the protein folds under physical principles, known as the protein folding problem, is one of the main tasks in modern biophysics. Coarse-grained methods play an increasingly important role in the simulation of protein folding, especially for large proteins. In recent years, we proposed a novel coarse-grained method derived from the topological soliton model, in terms of the backbone Cα chain. In this review, we will first systematically address the theoretical method of topological soliton. Then some successful applications will be displayed, including the thermodynamics simulation of protein folding, the property analysis of dynamic conformations, and the multi-scale simulation scheme. Finally, we will give a perspective on the development and application of topological soliton.

All-Atom Direct Folding Simulation for Proteins Using the Accelerated Molecular Dynamics in Implicit Solvent Model

We report the results of protein folding （219M, C34, N36, 2KES, 2KHK） by the method of accelerated molecular dynamics （aMD） at room temperature with the implicit solvent model. Starting from the linear structures, these proteins successfully fold to the native structure in a lO0-ns aMD simulation. In contrast, they are failed under the traditional MD simulation in the same simulation time. Then we find that the lowest root mean square deviations of helix structures from the native structures are 0.36 A, 0.63 A, 0.52 A, 1.1 A and 0.78 A. What is more, native contacts, cluster and free energy analyses show that the results of the aMD method are in accordance with the experiment very well. All analyses show that the aMD can accelerate the simulation process, thus we may apply it to the field of computer aided drug designs.

High Performance Hydrophobic Interaction Chromatography-A New Approach to Separate Intermediates of Protein Folding──Ⅰ.Separation of Intermediates of Urea-unfolded α-Amylase

Based on the different hydrophobicities of the intermediates of proteins the various conformational intermediates of the refolding of a-amylase originally denatured with 8.0 mol/L urea solution were separated with high performance hydrophobic interaction chromatography(HPHIC). Compared to the separation of the same intermediates with weak anion exchange chromatography and size-exclusion chromatography the result obtained with HPHIC is the best It would be expected that HPHIC may be a strongly potential tool to separate intermediates of some proteins which cannot be, or cannot completely be refolded by HPHIC.

Protein folding, protein homeostasis,and cancer

Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery.

Nanosecond-time-resolved infrared spectroscopic study of fast relaxation kinetics of protein folding by means of laser-induced temperature-jump

Elucidating the initial kinetics of folding pathways is critical to the understanding of the protein folding mechanism. Transient infrared spectroscopy has proved a powerful tool to probe the folding kinetics. Herein we report the construction of a nanosecond laser-induced temperature-jump （T-jump） technique coupled to a nanosecond timeresolved transient mid-infrared （mid-IR） spectrometer system capable of investigating the protein folding kinetics with a temporal resolution of 50 ns after deconvolution of the instrumental response function. The mid-IR source is a liquid N2 cooled CO laser covering a spectral range of 5.0μm （2000 cm^-1）-6.5μm （1540 cm^-1）. The heating pulse was generated by a high pressure H2 Raman shifter at wavelength of 1.9μm. The maximum temperature-jump could reach as high as 26±1℃. The fast folding/unfolding dynamics of cytochrome C was investigated by the constructed system, providing an example.

Protein structural codes and nucleation sites for protein folding

One of the long-standing controversial arguments in protein folding is Levinthal＇s paradox. We have recently proposed a new nucleation hypothesis and shown that the nucleation residues are the most conserved sequences in protein. To avoid the complicated effect of tertiary interactions, we limit our search for structural codes to the nucleation residues. Starting with the hypotheses of secondary structure nucleation and conservation of residues important for folding, we have analysed 762 folds classified as unique by SCOP. Segments of 17 residues around the top 20% conserved amino acids are analysed, resulting in approximately 100 clusters each for the main secondary structure classes of helix, sheet and coil. Helical clusters have the longest correlation range, coils the shortest （four residues）. Strong specific sequence-structure correlation is observed for coil but not for helix and sheet, suggesting a mapping relationship between the sequence and the structure for coil. We propose that the central sequences in these clusters form ＇structural codes＇, a useful basis set for identifying nucleation sites, protein fragments stable in isolation, and secondary structural patterns in proteins （particularly turns and loops）.

Quantum intelligence on protein folding pathways

We study the protein folding problem on the base of our quantum approach by considering the model of protein chain with nine amino-acid residues.We introduce the concept of distance space and its projections on a XY-plane,and two characteristic quantities,one is called compactness of protein structure and another is called probability ratio involving shortest path.The concept of shortest path enables us to reduce the 388×388 density matrix to a 2×2 one from which the von Neumann entropy reflecting certain quantum coherence feature is naturally defined.We observe the time evolution of average distance and compactness solved from the classical random walk and quantum walk,we also compare the features of the time-dependence of Shannon entropy and von Neumann entropy.All the results not only reveal the fast quantum folding time but also unveil the existence of quantum intelligence hidden behind in choosing protein folding pathways.

Folding nucleus and unfolding dynamics of protein 2GB1

The folding of many small proteins is kinetically a two-state process with one major free-energy barrier to overcome,which can be roughly regarded as the inverse process of unfolding.In this work,we first use a Gaussian network model to predict the folding nucleus corresponding to the major free-energy barrier of protein 2 GB1,and find that the folding nucleus is located in theβ-sheet domain.High-temperature molecular dynamics simulations are then used to investigate the unfolding process of 2 GB1.We draw free-energy surface from unfolding simulations,taking RMSD and contact number as reaction coordinates,which confirms that the folding of 2 GB1 is kinetically a two-state process.The comparison of the contact maps before and after the free energy barrier indicates that the transition from native to non-native structure of the protein is kinetically caused by the destruction of theβ-sheet domain,which manifests that the folding nucleus is indeed located in theβ-sheet domain.Moreover,the constrained MD simulation further confirms that the destruction of the secondary structures does not alter the topology of the protein retained by the folding nucleus.These results provide vital information for upcoming researchers to further understand protein folding in similar systems.