The oxidative folding pathway of the disulfide containing protein bovine pancreatic trypsin inhibitor (BPTI) was one of the first to be elucidated and has served as a basis for understanding the folding pathways of other proteins. During the oxidative folding of reduced BPTI, two intermediates (N' and N*) accumulate in significant amounts and act as kinetic traps. Both N' and N* bury their two remaining free thiols in their hydrophobic cores, which inhibits further oxidation. Historically, the rate limiting step was considered to be the intramolecular rearrangements of N' and N* to another intermediate with two free thiols, NSH. The two free thiols in NSH are solvent-exposed and easily oxidized to a disulfide, producing native protein (N). Nevertheless, our research using reduced BPTI indicated that the folding rate of N* to N was proportional to the concentration of added glutathione disulfide (GSSG), inconsistent with the slow intramolecular rearrangement of N* to NSH. To confirm our initial results, the intermediate N* was purified and refolded in the presence of GSSG. The conversion of N* to N was dependent upon the disulfide concentration and singly mixed disulfide N*(SG) was observed during folding. These results emphasize that the folding of N* can proceed via a growth type pathway, direct oxidation of the two remaining thiols in N* by an exogenous small molecule disulfide, such as GSSG, to form N. Folding of reduced BPTI via N* was performed under changing concentrations of GSSG and GSH as a function of time. The folding was improved dramatically in terms of rate and yield. Aromatic disulfides and thiols have been demonstrated to improve the folding efficiency of disulfide containing proteins including ribonuclease A (RNase A) and lysozyme. Herein, N* and N' were refolded in the presence of aromatic disulfides. Folding of the two kinetic traps with aromatic disulfides indicated that folding proceed via a growth type pathway. The singly and doubly mixed disulfide intermediates were observed during most folding reactions. The oxidative folding of reduced BPTI with aromatic disulfides and thiols were also investigated. Reduced BPTI can be folded to disulfide intermediates rapidly.

No description available.

In prokaryotes, protein synthesis and folding are often coupled, and the protein begins to fold from the N-terminus as it is being synthesized. It has been hypothesised that there could be kinetic coupling of the speed of translation and the folding, which means that an altered rate of synthesis can cause a possible misfolding of the protein. Testing this hypothesis will be impactful for protein misfolding diseases such as Alzheimer’s, Parkinson’s, Huntington’s etc., and also help in the study of the effect of synonymous, non-synonymous and rare codon changes on a protein. However, research works in this regard are far and few and none of them have been carried out in a homologous in vitro system. This project is an attempt to study the co-translational folding of Escherichia coli protein dihydro folate reductase (DHFR) using an E. coli reconstituted transcription/ translation system (RTTF) in vitro. The preparatory phase involves: preparation of UAG mutants of the DHFR DNA (for site-specific incorporation of fluorescent dyes), preparation of amber tRNAs which recognise the UAG codons, aminoacylation of the tRNAs and labelling the amino acids with fluorescent dyes. The experimental phase involves: incorporation of each of the fluorescent amino acids in the protein during in vitro synthesis in steady-state, observing incorporation of the same in stopped-flow spectrofluorimeter, attempting to observe fluorescent resonance energy transfer (FRET) between the two dyes due to co-translational folding. The preparatory and experimental phases were completed successfully, and it has been established that the amino acids with the fluorescent moieties can be incorporated site specifically in the mutant protein. The synthesis of the protein was observed using stopped-flow spectrometer for each of the fluorescent amino acids individually.  The synthesis of the mutants using two sets of dye pairs was also observed using a steady-state fluorimeter as well as stopped-flow spectrofluorimeter and the FRET between the two fluorophores was obtained. Although further experiments are required to fully validate and standardize this technique,  it will, even now,  aid in the study of the folding of proteins in a cell-free system.

The differences between refolding mechanisms of sperm whale apomyoglobin subsequent to three different unfolding conditions have been examined by atomistic level computer simulations. The three unfolding conditions used in this work are high-temperature, low temperature and low pH. The folding of this protein has been extensively studied experimentally, providing a large data base of folding parameters which can be probed using simulations. The crystal structure of sperm whale myoglobin was taken from Protein Data Bank, followed by the removal of the heme unit and a subsequent energy minimization was performed in order to generate the native apomyoblogin form. Thus, the native conformation of apomyoglobin utilized is the same in all the three different refolding simulations done in the present work. The differences are the way the initial unfolded conformations were obtained. The refolding trajectories were obtained at room temperature using the Stochastic Difference Equation in Length algorithm. The results reveal differences between the three refolding routes. In contrast to previous molecular simulations that modeled low pH denaturation, an extended intermediate with large helical content was not observed in the refolding simulations from the high-temperature unfolded state. Otherwise, a structural collapse occurs without formation of helices or native contacts. Once the protein structure is more compact (radius of gyration less than 18 angstroms) secondary and tertiary structures appear. The low pH simulations show some agreement with the low pH experimental data and previous molecular dynamics simulations, like formation of a conformation having radius of gyration around 20 angstroms and large helical content. And the refolding simulations after the low temperature unfolding present differences in the properties of apomyoglobin folding route, comparing to the other two previous conditions. The collapse of the protein during folding occurs later in the simulation when compared with high-temperature denaturing state, but earlier when compared to low pH simulations. These differences strongly suggest that a protein can follow different folding routes, depending on the nature and the structure of the unfolded state.

<div> The folding and unfolding processes of poly-protein has been tremendously studied recently. The poly-protein dynamics under an external force can play an important role in addressing the issue of the mechanics of muscle tissue. In this research, we use a single-molecule technique: magnetic tweezers to observe the dynamics of 8-mer poly-protein L under different loads applied and then in different Tris-buffered salines. Our result shows that more protein domains unfold as the force load becomes larger. At 6, 7 and 8 pN loads, the poly-protein is most likely to stay in state 1, 3 and 6 with 1, 3 and 6 domains unfolded, respectively according to the probability distribution. This can be well explained by our constructed free energy-related model. The fit results give protein L parameters of persistence length of 0.4 nm, contour length of 18.8 nm and the unfolding energy of 6.5 kT, all in reasonable ranges based on previously reported literature.</div><div> Besides, we also find the dependency of transition rate on force load and salt. The poly-protein has lower transition rate at high force than at low force due to the free energy tilting effect since high force extremely decreases the possibility of protein unfolding that results in a huge drop in the total number of folding and unfolding events. This inverse proportion effect can also be seen in different TRIS-buffered salines (TRIS-150mM NaCl, TRIS-1M NaCl, and TRIS-1M KCl,). We explore the effect of salt concentration, when the concentration of NaCl is increased, the transition rate increases while the probability distribution remains almost the same, indicating the protein unfolding barrier is lowered without altering the overall energy landscape. We attribute this to, first, the charge shielding effect that more interactions between ions and water molecules occur, causing fewer water molecules available to interact with the charged part of protein than before, and, second, more direct interactions of ions with protein that might affect the electrostatic-related transition rate. Considering the effect of salt type, the two 1M alkali metal-chloride salines are compared. We conclude that ions with larger size have less effect on transition rate because ions with smaller size (Na+) can create stronger bonds with water that increase the interference on the protein interaction with water and can easier penetrate into protein to directly interact with the protein.</div>

No description available.

Protein aggregation is deleterious to human health and detrimental to therapeutic shelf-life. The physical processes that induce aggregation are the same processes that drive productive folding reactions. As such, protein aggregation is a non-productive form of protein folding. To gain insight into the steps that serve as a partition between the folding and aggregation reactions, the folding mechanisms of several β-rich proteins with links to human disease or medicine were examined. In the ALS-linked protein, SOD1, a subpopulation of the unfolded ensemble is found to be a common source of both nonnative structure and frustrated folding. These behaviors are only observed upon the reduction of the intrinsic disulfide bond, indicating that this covalent interaction wards against aggregation. The nonnative structure presents an attractive target for the development of new therapeutic agents. In VH domains from therapeutic mAbs, the intramolecular disulfide bond protects against aggregation. However, it can also introduce complexity to the folding mechanism. This complexity is linked to the formation of a strained orientation of the disulfide bond. This strained orientation of the disulfide in certain VH domains is energetically unfavorable enough to disrupt the formation of the disulfide in the full length mAbs. The novel relationship observed between disulfide orientation, folding complexity, and incomplete oxidation warrants further examination in other Ig domains. Overall, these results demonstrate that mapping the folding free energy landscape for proteins with roles in human disease or therapeutics can provide valuable insights for developing and improving treatment options.

(βα)8 TIM barrel proteins are one of the most common structural motifs found in biology. They have a complex folding free energy landscape that includes an initial off-pathway intermediate as well as two on-pathway intermediates. The formation of these intermediates is hypothesized to be driven by large clusters of the branched chain amino acids, isoleucine, leucine, and valine (ILV). All-atom MD simulations and circular dichroism experiments on polar mutants of the hydrophobic clusters of α-Trp synthase, a TIM barrel protein, revealed the importance of dehydrating the clusters on intermediate states. Custom, single-piece microfluidic chips were interfaced with small angle x-ray scattering and time resolved FRET experiments to monitor the role of a large ILV cluster on the microsecond timescale in a second TIM barrel protein, sIGPS. Dimensional analysis of the initial misfolded intermediate showed an ILV cluster was responsible for the initiation of structure in the intermediate. Early structure formation in the ILV cluster was confirmed by coarse grained simulations. Native state hydrogen exchange experiments were used to probe the higher energy species that are in equilibrium with the native state. Results from the NMR experiment complement the kinetic studies as the core of stability found by NMR mapped back to the same region of the ILV cluster that was found to initiate folding. When taken together, the results show the importance of hydrophobic clusters on the entire free energy surface of TIM barrel proteins.

Early events in folding can determine if a protein is going to fold, misfold, or aggregate. Understanding these deterministic events is paramount for de novo protein engineering, the enhancement of biopharmaceutical stabilities, and understanding neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. However, the physicochemical and structural biases within high energy states of protein biopolymers are poorly understood. A combined experimental and computational study was conducted on the small β/α-repeat protein CheY to determine the structural basis of its submillisecond misfolding reaction to an off-pathway intermediate. Using permutations, we were able to discriminate between the roles of two proposed mechanisms of folding; a nucleation condensation model, and a hydrophobic collapse model driven by the formation of clusters of isoleucine, leucine, and valine (ILV) residues. We found that by altering the ILV cluster connectivity we could bias the early folding events to either favor on or off-pathway intermediates. Structural biases were also experimentally observed in the unfolded state of a de novo designed synthetic β/α-repeat protein, Di-III_14. Although thermodynamically and kinetically 2-state, Di-III_14 has a well structured unfolded state that is only observable under native-favoring conditions. This unfolded state appears to retain native-like structure, consisting of a hydrophobic 7 core (69% ILV) stabilized by solvent exposed polar groups and long range electrostatic interactions. Together, these results suggest that early folding events are largely deterministic in these two systems. Generally, low contact order ILV clusters favor local compaction and, in specific cases, long range electrostatic interactions may have stabilizing effects in higher energy states.

Sequence divergence of orthologous proteins enables adaptation to a plethora of environmental stresses and promotes evolution of novel functions. As one of the most common motifs in biology capable of diverse enzymatic functions, the TIM barrel represents an ideal model system for mapping the phenotypic manifestations of protein sequence. Limits on evolution imposed by constraints on sequence and structure were investigated using a model TIM barrel protein, indole-3-glycerol phosphate synthase (IGPS). Exploration of fitness landscapes of phylogenetically distant orthologs provides a strategy for elucidating the complex interrelationship in the context of a protein fold. Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Significant correlations between the fitness landscapes of distant orthologues implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape. These results suggest that fitness landscapes of point mutants can be successfully translocated in sequence space, where knowledge of one landscape may be predictive for the landscape of another ortholog. Analysis of a surprising class of beneficial mutations in all three IGPS orthologs pointed to a long-range allosteric pathway towards the active site of the protein. Biophysical and biochemical analyses provided insights into the molecular mechanism of these beneficial fitness effects. Epistatic interactions suggest that the helical shell may be involved in the observed allostery. Taken together, knowledge of the fundamental properties of the TIM protein architecture will provide new strategies for de novo protein design of a highly targeted protein fold.