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.

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.

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.

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.

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.

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.

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.

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）.

Quasi-physical Algorithm for Protein Folding in an Off-Lattice Model

We study a three-dimensional off-lattice protein folding model, which involves two species of residues interacting through Lennard-Jones potentials. By incorporating an extra energy contribution into the original potential function, we replace the original constrained problem with an unconstrained minimization of a mixed potential function. As such an efficient quasi-physical algorithm for solving the protein folding problem is presented. We apply the proposed algorithm to sequences with up to 55 residues and compare the computational results with the putative lowest energy found by several of the most famous algorithms, showing the advantages of our method. The dynamic behavior of the quasi-physlcal algorithm is also discussed.

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.

Advances in the study of protein folding and endoplasmic reticulum-associated degradation in mammal cells

The endoplasmic reticulum is a key site for protein production and quality control.More than one-third of proteins are synthesized and folded into the correct three-dimensional conformation in the endoplasmic reticulum.However,during protein folding,unfolded and/or misfolded proteins are prone to occur,which may lead to endoplasmic reticulum stress.Organisms can monitor the quality of the proteins produced by endoplasmic reticulum quality control(ERQC)and endoplasmic reticulum-associated degradation(ERAD),which maintain endoplasmic reticulum protein homeostasis by degrading abnormally folded proteins.The underlying mechanisms of protein folding and ERAD in mammals have not yet been fully explored.Therefore,this paper reviews the process and function of protein folding and ERAD in mammalian cells,in order to help clinicians better understand the mechanism of ERAD and to provide a scientific reference for the treatment of diseases caused by abnormal ERAD.

Electrostatic and hydrophobic interaction cooperative nanochaperone regulates protein folding

Natural molecular chaperones utilize spatially ordered multiple molecular forces to effectively regulate protein folding.However,synthesis of such molecules is a big challenge.The concept of“aggregate science”provides insights to construct chemical entities(aggregates)beyond molecular levels to mimic both the structure and function of natural chaperone.Inspired by this concept,herein we fabricate a novel multi-interaction(i.e.,electrostatic and hydrophobic interaction)cooperative nanochaperone(multi-co-nChap)to regulating protein folding.This multi-co-nChap is fabricated by rationally introducing electrostatic interactions to the surface(corona)and confined hydrophobic microdomains(shell)of traditional single-hydrophobic interaction nanochaperone.We demonstrate that the corona electrostatic attraction facilitates the diffusion of clients into the hydrophobic microdomains,while the shell electrostatic interaction balances the capture and release of clients.By finely synergizing corona electrostatic attraction with shell electrostatic repulsion and hydrophobic interaction,the optimized multi-co-nChap effectively facilitated de novo folding of nascent polypeptides.Moreover,the synergy between corona electrostatic attraction,shell electrostatic attraction and shell hydrophobic interaction significantly enhanced the capability of multi-co-nChap to protect native proteins from denaturation at harsh temperatures.This work provides important insights for understanding and design of nanochaperone,which is a kind of ordered aggregate with chaperone-like activity that beyond the level of single molecule.

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.

Protein folding as a quantum transition between conformational states

Assuming that the main variables in the life processes at the molecular level are the conforma- tion of biological macromolecules and their frontier electrons a formalism of quantum theory on conformation-electron system is proposed. Based on the quantum theory of conformation-electron system, the protein folding is regarded as a quantum transition between torsion states on polypep- tide chain, and the folding rate is calculated by nonadiabatic operator method. The rate calculation is generalized to the case of frequency variation in folding. An analytical form of protein folding rate formula is obtained, which can be served as a useful tool for further studying protein folding. The application of the rate theory to explain the protein folding experiments is briefly summarized. It includes the inertial moment dependence of folding rate, the unified description of two-state and multistate protein folding, the relationship of folding and unfolding rates versus denaturant concen- tration, the distinction between exergonic and endergonic foldings, the ultrafast and the downhill folding viewed from quantum folding theory, and, finally, the temperature dependence of folding rate and the interpretation of its non-Arrhenius behaviors. All these studies support the view that the protein folding is essentially a quantum transition between conformational states.

The dynamical contact order:Protein folding rate parameters based on quantum conformational transitions

Protein folding is regarded as a quantum transition between the torsion states of a polypeptide chain.According to the quantum theory of conformational dynamics,we propose the dynamical contact order(DCO) defined as a characteristic of the contact described by the moment of inertia and the torsion potential energy of the polypeptide chain between contact residues.Conse-quently,the protein folding rate can be quantitatively studied from the point of view of dynamics.By comparing theoretical calculations and experimental data on the folding rate of 80 proteins,we successfully validate the view that protein folding is a quantum conformational transition.We conclude that(i) a correlation between the protein folding rate and the contact inertial moment exists;(ii) multi-state protein folding can be regarded as a quantum conformational transition similar to that of two-state proteins but with an intermediate delay.We have estimated the order of magnitude of the time delay;(iii) folding can be classified into two types,exergonic and endergonic.Most of the two-state proteins with higher folding rate are exergonic and most of the multi-state proteins with low folding rate are endergonic.The folding speed limit is determined by exergonic folding.

Quantum theory on protein folding

The conformational change of biological macromolecule is investigated from the point of quantum transition.A quantum theory on protein folding is proposed.Compared with other dynamical variables such as mobile electrons,chemical bonds and stretching-bending vibrations the molecular torsion has the lowest energy and can be looked as the slow variable of the system.Simultaneously,from the multi-minima property of torsion potential the local conformational states are well defined.Following the idea that the slow variables slave the fast ones and using the nonadiabaticity operator method we deduce the Hamiltonian describing conformational change.It is shown that the influence of fast variables on the macromolecule can fully be taken into account through a phase transformation of slow variable wave function.Starting from the conformation-transition Hamiltonian the nonradiative matrix element was calculated and a general formulas for protein folding rate was deduced.The analytical form of the formula was utilized to study the temperature dependence of protein folding rate and the curious non-Arrhenius temperature relation was interpreted.By using temperature dependence data the multi-torsion correlation was studied.The decoherence time of quantum torsion state is estimated.The proposed folding rate formula gives a unifying approach for the study of a large class problems of biological conformational change.

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.

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.

Stabilities and Dynamics of Protein Folding Nuclei by Molecular Dynamics Simulation

To understand how the stabilities of key nuclei fragments affect protein folding dynamics, we simulate by molecular dynamics （MD） simulation in aqueous solution four fragments cut out of a protein G, including one a-helix （seqB： KVFKQYAN）, two -turns （seqA： LNGKTLKG and seqC： YDDATKTF）, and one -strand （seqD： DGEWTYDD）. The Markov State Model clustering method combined with the coarse-grained conformation letters method are employed to analyze the data sampled from 2-#s equilibrium MD simulation trajectories. We find that seqA and seqB have more stable structures than their native structures which become metastable when cut out of the protein structure. As expected, seqD alone is flexible and does not have a stable structure. Throughout our simulations, the native structure of seqC is stable but cannot be reached if starting from a structure other than the native one, implying a funnel-shape free energy landscape of seqC in aqueous solution. All the above results suggest that different nuclei have different formation dynamics during protein folding, which may have a major contribution to the hierarchy of protein folding dynamics.