C. Robert Matthews

The mechanism of protein folding

Abstract Recent advances in spectroscopy, peptide synthesis, and protein engineering have provided new evidence on the mechanisms of protein folding which supports the hypothesis that there are some events common to the early, middle and late stages of folding. By focusing on these common events rather than the specific differences, the essential aspects of the mechanisms by which the amino acid sequence of a protein directs its rapid and efficient folding to yield the native conformation can be highlighted.

Multistate Equilibrium Unfolding of Escherichia coli Dihydrofolate Reductase: Thermodynamic and Spectroscopic Description of the Native, Intermediate, and Unfolded Ensembles†

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

The earliest kinetic folding events for (βα)8 barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (βα)8 barrel protein. Within 150 μs, the α-subunit of Trp synthase (αTS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 μs, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase.

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The approximately 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this beta-barrel protein.

Barriers in protein folding reactions.

Publisher Summary This chapter focuses on the experimental thermodynamic and structural characterization of the barriers in the folding of single-domain globular proteins. The chapter also focuses on the structural aspects of the transition state of folding reactions, primarily, on mutational analyses and other protein engineering studies in which folding and/or unfolding kinetics have been investigated. The thermodynamic properties and the dynamics of the barrier crossings are also discussed. Studies of the elementary events in protein folding through simple model systems and a comparative overview of insights obtained from experimental and computational results are presented in the chapter. Many single-domain proteins, typically composed of 300 or fewer amino acids, exhibit intermediates in their folding reactions. Studies in which the properties of the intermediates have been the focus, as opposed to the transition states, are discussed. The chapter concludes that the merging of computational and experimental studies offers the prospect of unprecedented insight into the structure and dynamics of barriers in protein folding reactions.

A microchannel solution mixer for studying microsecond protein folding reactions

Many proteins fold through intermediates that are populated in the submillisecond time regime. To monitor directly the formation of these kinetic intermediates, we have developed a simple, robust, easy to assemble continuous flow mixer for studying folding reactions in the 35–1000μs time regime. The mixer is constructed by laser-machining 75-μm channels in a 127-μm-thick polyimide or polyetheretherketone polymer wafer. Mixing times of ∼25to∼50μs can be achieved for a 1∕10 dilution reaction of 8M urea with flow rates of 10–20mL∕min. CCD-based steady-state and time-correlated single-photon-counting-based fluorescence detection strategies are described. Preliminary results on the early events in the refolding of cytochrome c are presented.

Sequential vs. parallel protein-folding mechanisms: experimental tests for complex folding reactions.

The recent emphasis on rough energy landscapes for protein folding reactions by theoreticians, and the many observations of complex folding kinetics by experimentalists provide a rationale for a brief literature survey of various empirical approaches for validating the underlying mechanisms. The determination of the folding mechanism is a key step in defining the energy surface on which the folding reactions occurs and in interpreting the effects of amino acid replacements on this reaction. Case studies that illustrate methods for differentiating between sequential and parallel channel folding mechanisms are presented. The ultimate goal of such efforts is to understand how the one-dimensional information contained in the amino acid sequence is rapidly and efficiently translated into three-dimensional structure.

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu2+, even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.

Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: A test of the conservation of folding mechanisms hypothesis in (βα)8 barrels

Abstract As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli . A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed. Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in αTS in terms of its thermodynamic properties and secondary structure. Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase ( via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate. At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism. The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species. Comparison of the results for sIGPS and αTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events. The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins.

Minireview: structural insights into early folding events using continuous-flow time-resolved small-angle X-ray scattering.

Small-angle X-ray scattering (SAXS) is a powerful method for obtaining quantitative structural information on the size and shape of proteins, and it is increasingly used in kinetic studies of folding and association reactions. In this minireview, we discuss recent developments in using SAXS to obtain structural information on the unfolded ensemble and early folding intermediates of proteins using continuous-flow mixing devices. Interfacing of these micromachined devices to SAXS beamlines has allowed access to the microsecond time regime. The experimental constraints in implementation of turbulence and laminar flow-based mixers with SAXS detection and a comparison of the two approaches are presented. Current improvements and future prospects of microsecond time-resolved SAXS and the synergy with ab initio structure prediction and molecular dynamics simulations are discussed.