Folding and Aggregation Are Selectively Influenced by the Conformational Preferences of the α-Helices of Muscle Acylphosphatase
49
Citation
38
Reference
10
Related Paper
Citation Trend
Abstract:
The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.Keywords:
Helix (gastropod)
Folding (DSP implementation)
Alpha helix
Cells are constantly subjected to stresses; these stresses take the form of heat, heavy metals, metabolic poisons, non-native peptides, and many others. All of these stresses have the potential to cause protein misfolding, which drives protein aggregation. In neurons, long-term protein misfolding and aggregation is known to lead to the onset of neurodegenerative diseases such as Parkinson's, Huntington's, Alzheimer's, ALS, Scrapie, and others. The cellular response to stress and protein misfolding employs the mol. chaperones. In this presentation, we will explore the behavior of protein misfolding and aggregation in the presence of mol. chaperones. In line with exptl. results that show discontinuous jumps in aggregate concn. with subtle changes in protein concns. and cellular parameters, we demonstrate that simple models show the early stage of protein aggregation, sol. oligomer formation, is a bistable process that depends on the local concn. of mol. chaperones. Understanding the formation of the sol. oligomers is essential since many recent exptl. studies have implicated these species as the proteotoxic species in neurodegenerative disease. [on SciFinder (R)]
Chaperone (clinical)
Oligomer
Chemical chaperone
Bistability
Cite
Citations (0)
Cellular protein folding is challenged by environmental stress and aging, which lead to aberrant protein conformations and aggregation. One way to antagonize the detrimental consequences of protein misfolding is to reactivate vital proteins from aggregates. In the yeast Saccharomyces cerevisiae, Hsp104 facilitates disaggregation and reactivates aggregated proteins with assistance from Hsp70 (Ssa1) and Hsp40 (Ydj1). The small heat shock proteins, Hsp26 and Hsp42, also function in the recovery of misfolded proteins and prevent aggregation in vitro, but their in vivo roles in protein homeostasis remain elusive. We observed that after a sublethal heat shock, a majority of Hsp26 becomes insoluble. Its return to the soluble state during recovery depends on the presence of Hsp104. Further, cells lacking Hsp26 are impaired in the disaggregation of an easily assayed heat-aggregated reporter protein, luciferase. In vitro, Hsp104, Ssa1, and Ydj1 reactivate luciferase:Hsp26 co-aggregates 20-fold more efficiently than luciferase aggregates alone. Small Hsps also facilitate the Hsp104-mediated solubilization of polyglutamine in yeast. Thus, Hsp26 renders aggregates more accessible to Hsp104/Ssa1/Ydj1. Small Hsps partially suppress toxicity, even in the absence of Hsp104, potentially by sequestering polyglutamine from toxic interactions with other proteins. Hence, Hsp26 plays an important role in pathways that defend cells against environmental stress and the types of protein misfolding seen in neurodegenerative disease.
Chaperone (clinical)
Cite
Citations (284)
Helix (gastropod)
Electrostatics
Alpha helix
Static electricity
Cite
Citations (56)
Folding (DSP implementation)
Globular protein
Cite
Citations (55)
Proteins are delicate, versatile and structurally complex biomolecules which regulate fundamental processes of the cellular systems. They are synthesized as long stretches of amino acid chains and in order to achieve the functional state each polypeptide chain must be folded into unique 3-dimensional structure [1] . However, proteins have a very narrow range of thermodynamically stable physiological environment inside the cells to achieve correct folding and to function [2] .. In addition, several chronic challenges such as aging related physiological changes, diseases and certain stress conditions also interfere with protein functioning [2] . Therefore, how the cells manage their proteome and ensure their metastable conformations to retain the conformational flexibility in the ever changing cellular environment has emerged as a fundamental question in contemporary biomedical research.
Chaperone (clinical)
Co-chaperone
Folding (DSP implementation)
Cite
Citations (4)
Helix (gastropod)
Alpha helix
Alpha (finance)
Cite
Citations (114)
The pathway of protein folding is now being analyzed at the resolution of individual residues by kinetic measurements on suitably engineered mutants. The kinetic methods generally employed for studying folding are typically limited to the time range of > or = 1 ms because the folding of denatured proteins is usually initiated by mixing them with buffers that favor folding, and the dead time of rapid mixing experiments is about a millisecond. We now show that the study of protein folding may be extended to the microsecond time region by using temperature-jump measurements on the cold-unfolded state of a suitable protein. We are able to detect early events in the folding of mutants of barstar, the polypeptide inhibitor of barnase. A preliminary characterization of the fast phase from spectroscopic and phi-value analysis indicates that it is a transition between two relatively solvent-exposed states with little consolidation of structure.
Barnase
Microsecond
Contact order
Folding (DSP implementation)
Cite
Citations (136)
Contact order
Folding (DSP implementation)
Equilibrium unfolding
Cite
Citations (1,515)
Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to “zipper up,” while having an extrapolated folding rate of k f = 2 × 10 5 s -1 . These properties make it likely to attain the elusive “downhill folding” limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. The stability and its denaturant dependence for the hydrogen bonds in the central part of protein equal the values calculated for whole-molecule unfolding. Like-wise, their closing and opening rates indicate that these hydrogen bonds are broken and reformed in a single cooperative event representing the folding transition from the fully unfolded state to the native state. Additionally, closing rates for these hydrogen bonds agree with the extrapolated barrier-limited folding rate observed near the melting transition. Therefore, even in the absence of denaturant, where Δ G eq ≈ -6 kcal·mol -1 (1 cal = 4.18 J) and τ f ≈ 6 μs, folding remains cooperative and barrier-limited. Given that this prime candidate for downhill folding fails to do so, we propose that protein folding will remain barrier-limited for proteins that fold cooperatively.
Folding (DSP implementation)
Contact order
Cite
Citations (26)
Protein aggregation can be defined as the sacrifice of stabilizing intrachain contacts of the functional state that are replaced with interchain contacts to form non-functional states. The resulting aggregate morphologies range from amorphous structures without long-range order typical of nondisease proteins involved in inclusion bodies to highly structured fibril assemblies typical of amyloid disease proteins. In this Account, we describe the development and application of computational models for the investigation of nondisease and disease protein aggregation as illustrated for the proteins L and G and the Alzheimer's Aβ systems. In each case, we validate the models against relevant experimental observables and then expand on the experimental window to better elucidate the link between molecular properties and aggregation outcomes. Our studies show that each class of protein exhibits distinct aggregation mechanisms that are dependent on protein sequence, protein concentration, and solution conditions. Nondisease proteins can have native structural elements in the denatured state ensemble or rapidly form early folding intermediates, which offers avenues of protection against aggregation even at relatively high concentrations. The possibility that early folding intermediates may be evolutionarily selected for their protective role against unwanted aggregation could be a useful strategy for reengineering sequences to slow aggregation and increase folding yield in industrial protein production. The observed oligomeric aggregates that we see for nondisease proteins L and G may represent the nuclei for larger aggregates, not just for large amorphous inclusion bodies, but potentially as the seeds of ordered fibrillar aggregates, since most nondisease proteins can form amyloid fibrils under conditions that destabilize the native state. By contrast, amyloidogenic protein sequences such as Aβ1−40,42 and the familial Alzheimer's disease (FAD) mutants favor aggregation into ordered fibrils once the free-energy barrier for forming a critical nucleus is crossed. However, the structural characteristics and oligomer size of the soluble nucleation species have yet to be determined experimentally for any disease peptide sequence, and the molecular mechanism of polymerization that eventually delineates a mature fibril is unknown. This is in part due to the limited experimental access to very low peptide concentrations that are required to characterize these early aggregation events, providing an opportunity for theoretical studies to bridge the gap between the monomer and fibril end points and to develop testable hypotheses. Our model shows that Aβ1−40 requires as few as 6−10 monomer chains (depending on sequence) to begin manifesting the cross-β order that is a signature of formation of amyloid filaments or fibrils assessed in dye-binding kinetic assays. The richness of the oligomeric structures and viable filament and fibril polymorphs that we observe may offer structural clues to disease virulence variations that are seen for the WT and hereditary mutants.
Folding (DSP implementation)
Amyloid (mycology)
Cite
Citations (36)