Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.     

Characterization of the hydrodynamic properties of the folding transition state of an SH3 domain by magnetization transfer NMR spectroscopy

Protein folding kinetic data have been obtained for the marginally stable N-terminal Src homology 3 domain of the Drosophila protein drk (drkN SH3) in an investigation of the hydrodynamic properties of its folding transition state. Due to the presence of NMR resonances of both folded and unfolded states at equilibrium, kinetic data can be derived from NMR magnetization transfer techniques under equilibrium conditions. Kinetic analysis as a function of urea (less than approximately 1 M) and glycerol enables determination of alpha values, measures of the energetic sensitivity of the transition state to the perturbation relative to the end states of the protein folding reaction (the folded and unfolded states). Both end states have previously been studied experimentally by NMR spectroscopic and other biophysical methods in great detail and under nondenaturing conditions. Combining these results with the kinetic folding data obtained here, we can characterize the folding transition state without requiring empirical models for the unfolded state structure. We are thus able to give a reliable measure of the solvent-accessible surface area of the transition state of the drkN SH3 domain (4730 +/- 360 A(2)) based on urea titration data. Glycerol titration data give similar results and additionally demonstrate that folding of this SH3 domain is dependent on solvent viscosity, which is indicative of at least partial hydration of the transition state. Because SH3 domains appear to fold by a common folding mechanism, the data presented here provide valuable insight into the transition states of the drkN and other SH3 domains.     

Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding

Applications of sensitive new technologies, in particular, two-dimensional NMR spectroscopy, have allowed detection of folded structures in short peptide fragments of proteins in aqueous solution under conditions where native proteins fold. These structures are in rapid dynamic exchange with unfolded states. These observations provide evidence in support of models for protein folding which postulate localized regions of folded structure as initiation sites for the folding process. Since these initiation processes are expected to be rapid, such models are consistent with kinetic evidence that the rate-determining steps of protein folding occur late in the process and probably involve rearrangement of incorrectly folded intermediates.     

Denatured collapsed states in protein folding: example of apomyoglobin.

Experimental approaches, including circular dichroism, small angle X-ray scattering, steady-state fluorescence, and fluorescence energy transfer, were applied to study the 3D-structure of apomyolgobin in different conformational states. These included the native and molten globules, along with either less ordered conformations induced by the addition of anions or completely unfolded states. The results show that the partially folded forms of apomyoglobin stabilized by KCl and/or Na(2)SO(4) under unfolding conditions (pH 2) exhibit a significant amount of secondary structure (circular dichroism), low packing density of protein molecules (SAXS), and native-like dimensions of the AGH core (fluorescence energy transfer). This finding indicates that a native-like tertiary fold of the polypeptide chain, i.e., the spatial organization of secondary structure elements, most likely emerges prior to the formation of the molten globule state.     

Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin

Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions. The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 1.5 for lysozyme and pH 7.0 for alpha-lactalbumin at 4.5 degrees C. In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically. The major fast phase of lysozyme folding has a decay time of 15 s. The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein. In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements. This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands. Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate. Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH-jump-induced folding and unfolding studies of barstar: evidence for multiple folding and unfolding pathways.

Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.     

Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

Acid- and pressure-induced (un)folding of yeast glutathione reductase: competition between protein oligomerization and aggregation

Glutathione reductase (GR) is a homodimeric flavoenzyme involved in cellular defense against oxidative stress. In the present study, we have used a combination of acidic pH and hydrostatic pressure to investigate the (un)folding transition of yeast GR. Our results indicate that at pH 2 a distinct partially folded state is stabilized, as judged by intrinsic fluorescence, bis ANS binding and circular dichroism (CD) analysis. Further characterization of this partially folded state by size exclusion chromatography revealed that it corresponds to expanded GR monomers. CD analysis at pH 2 showed a significant loss of secondary structure. The partially folded GR monomers stabilized at pH 2 were fully and reversibly unfolded using hydrostatic pressure (up to 3.5kbar) as a thermodynamic perturbant. By contrast, return to physiological pH after exposure to acidic pH led to a competing reaction between refolding dimerization and aggregation of GR. These results support the notion that a partially folded intermediate state is not only critical for folding of GR but also appears to be a seed for protein aggregation.     

Concerted folding and binding of a flexible colicin domain to its periplasmic receptor TolA

Compared with folded structures, natively unfolded protein domains are over-represented in protein-protein and protein-DNA interactions. Such domains are common features of all colicins and are required for their translocation across the outer membrane of the target Escherichia coli cell. All of these domains bind to at least one periplasmic protein of the Tol or Ton family. Similar domains are found in Ton-dependent outer membrane transporters, indicating they may interact in a related manner. In this article we have studied binding of the colicin N translocation domain to its periplasmic receptor TolA, by fluorescence resonance energy transfer (FRET) using fluorescent probes attached to engineered cysteine residues and NMR techniques. The domain exhibits a random coil circular dichroism spectrum. However, FRET revealed that guanidinium hydrochloride denaturation caused increases in all measured intramolecular distances showing that, although natively unfolded, the domain is not extended. Furthermore NMR reported a compact hydrodynamic radius of 18 A. Nevertheless the FRET-derived distances changed upon binding to TolA indicating a significant structural rearrangement. Using 1H-15N NMR we show that, when bound, the peptide switches from a disordered state to an ordered state. The kinetics of binding and the associated structural change were measured by stopped-flow methods, and both events appear to occur simultaneously. The data therefore suggest that this molecular recognition involves the concerted binding and folding of a flexible but collapsed state.     

The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H

Elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. Here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the Thermus thermophilus ribonuclease H, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an α-helix and two β-strands (51 folded residues). Using multidimensional NMR, we solved the structure of this intermediate mimic to an atomic resolution (backbone rmsd, 0.51 Å). It has a native-like backbone topology and shows some local deviations from the native structure, revealing that the structure of the folded region of an early folding intermediate can be as well defined as the native structure. The topological parameters calculated from the structures of the intermediate mimic and the native state predict that the intermediate should fold on a millisecond time scale or less and form much faster than the native state. Other factors that may lead to the slow folding of the native state and the accumulation of the intermediate before the rate-limiting transition state are also discussed.     

Role of ligand substitution in ferrocytochrome c folding

The ligand substitutions that occur during the folding of ferrocytochrome c [Fe(II)cyt c] have been monitored by transient absorption spectroscopy. The folding reaction was triggered by photoinduced electron transfer to unfolded Fe(III)cyt c in guanidine hydrochloride (GuHCl) solutions. Assignments of ligation states were made by reference to the spectra of the imidazole and methionine adducts of N-acetylated microperoxidase 8. At pH 7, the heme in unfolded Fe(II)cyt c is ligated by native His18 and HisX (X = 26, 33) residues. The native Met80 ligand displaces HisX only in the last stages of folding. The ferroheme is predominantly five-coordinate in acidic solution; it remains five-coordinate until the native methionine binds the heme to give the folded protein (the rate of the methionine binding step is 16 +/- 5 s-1 at pH 5, 3.2 M GuHCl). The evidence suggests that the substitution of histidine by methionine is strongly coupled to backbone folding.     

Kinetic analysis of R67 dihydrofolate reductase folding: from the unfolded monomer to the native tetramer

R67 dihydrofolate reductase (DHFR) is a homotetrameric enzyme. Its subunit has a core structure consisting of five antiparallel beta-strands that form a compact beta-barrel. Our interest was to describe the molecular mechanism of the complete folding pathway of this beta-sheet protein, focusing on how the oligomerization steps are coordinated with the formation of secondary and tertiary structures all along the folding process. The folding kinetics of R67 dihydrofolate reductase into dimers at pH 5.0 were first examined by intrinsic tryptophan fluorescence, fluorescence energy transfer, and circular dichroism spectroscopy. The process was shown to consist of at least four steps, including a burst, a rapid, a medium, and a slow phase. Measurements of the ellipticity at 222 nm indicated that about 50% of the total change associated with refolding occurred during the 4 ms dead time of the stopped-flow instrument, indicating a substantial burst of secondary structure. The bimolecular association step was detected using fluorescence energy transfer and corresponded to the rapid phase. The slow phase was attributed to a rate-limiting isomerization of peptidyl-prolyl bonds involving 15% of the unfolded population. A complete folding pathway from the unfolded monomer to the native tetramer was proposed and an original model based upon the existence of early partially folded monomeric intermediates, rapidly stabilized in a dimeric form able to self-associate into the native homotetramer was formulated. The rate constants of these various steps were determined by fitting the kinetic traces to this model and supported our mechanistic assumptions.     

pH jump studies of the folding of the multidomain ribosomal protein L9: the structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain

The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein.     

pH-dependent urea-induced unfolding of stem bromelain: Unusual stability against urea at neutral pH

Equilibrium unfolding of stem bromelain (SB) with urea as a denaturant has been monitored as a function of pH using circular dichroism and fluorescence emission spectroscopy. Urea-induced denaturation studies at pH 4.5 showed that SB unfolds through a two-state mechanism and yields ΔG (free energy difference between the fully folded and unfolded forms) of ∼5.0 kcal/mol and C _m (midpoint of the unfolding transition) of ∼6.5 M at 25°C. Very high concentration of urea (9.5 M) provides unusual stability to the protein with no more structural loss and transition to a completely unfolded state.     

Apoflavodoxin (un)folding followed at the residue level by NMR

The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H-15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H-15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.     

Effect of backbone cyclization on protein folding stability: chain entropies of both the unfolded and the folded states are restricted

Circular versions of a large number of proteins have been designed by connecting the N and C termini via peptide linkers. A motivation for these designs is the assumed enhancement in folding stability, because backbone cyclization reduces the chain entropy of the unfolded state. Here, it is recognized that backbone cyclization also reduces the chain entropy of a flexible peptide linker in the folded state. Specifically, the end-to-end distance of the linker is restricted to fluctuations around the average displacement between the N and C termini of the folded protein. The balance of the chain-entropy reductions in the folded and unfolded states is used to predict the change in the unfolding free energy, deltadeltaG(cycl), by backbone cyclization. Predicted values of deltadeltaG(cycl) are in quantitative agreement with results of a careful study on cyclizing the 34 residue PIN1 WW domain by linkers with two to seen residues. The experimental results of an optimal linker length l=4 and a maximum stabilization of 1.7 kcal/mol are reproduced. Calculations of deltadeltaG(cycl) for a broad selection of circular proteins suggest that the stabilizing effect of backbone cyclization is modest, reflecting entropy reductions in both the unfolded and the folded states.     

Conformational stability of apoflavodoxin.

Flavodoxins are alpha/beta proteins that mediate electron transfer reactions. The conformational stability of apoflavodoxin from Anaboena PCC 7119 has been studied by calorimetry and urea denaturation as a function of pH and ionic strength. At pH > 12, the protein is unfolded. Between pH 11 and pH 6, the apoprotein is folded properly as judged from near-ultraviolet (UV) circular dichroism (CD) and high-field 1H NMR spectra. In this pH interval, apoflavodoxin is a monomer and its unfolding by urea or temperature follows a simple two-state mechanism. The specific heat capacity of unfolding for this native conformation is unusually low. Near its isoelectric point (3.9), the protein is highly insoluble. At lower pH values (pH 3.5-2.0), apoflavodoxin adopts a conformation with the properties of a molten globule. Although apoflavodoxin at pH 2 unfolds cooperatively with urea in a reversible fashion and the fluorescence and far-UV CD unfolding curves coincide, the transition midpoint depends on the concentration of protein, ruling out a simple two-state process at acidic pH. Apoflavodoxin constitutes a promising system for the analysis of the stability and folding of alpha/beta proteins and for the study of the interaction between apoflavoproteins and their corresponding redox cofactors.     

A conformational analysis of leucine enkephalin as a function of pH

The conformations of Leu enkephalin in aqueous solution have been investigated as a function of pH using molecular dynamics simulations. The simulations suggest the peptide backbone exists as a mixture of folded and unfolded forms (approximately 50% each) at neutral pH, but is always unfolded at low or high pH. The folded form at neutral pH possesses a 2 --> 5 hydrogen bond and a close head to tail separation. No significant intramolecular hydrogen bonding of the carbonyl oxygens was observed in either the folded or unfolded forms of the peptide. Analysis of the Gly carbonyl oxygens and terminal groups indicated that, while the conformational population distribution of Leu enkephalin did vary noticeably as a function of pH, their hydration was essentially independent of pH and in agreement with the available NMR data. Further study indicated that the unfolded state of the peptide was not random in nature and consisted of one major unfolded backbone arrangement stabilized by a persistent hydrophobic interaction between the side chains of Tyr and Leu.     

Temperature-dependent beta-sheet formation in beta-amyloid Abeta(1-40) peptide in water: uncoupling beta-structure folding from aggregation

To probe the role of temperature in the conversion of soluble Alzheimer's beta-amyloid peptide (Abeta) to insoluble beta-sheet rich aggregates, we analyzed the solution conformation of Abeta(1-40) from 0 to 98 degrees C by far-UV circular dichroism (CD) and native gel electrophoresis. The CD spectra of 15-300 microg/ml Abeta(1-40) in aqueous solution (pH approximately 4.6) at 0 degrees C are concentration-independent and suggest a substantially unfolded and/or unusually folded conformation characteristic of Abeta monomer or dimer. Heating from 0 to 37 degrees C induces a rapid reversible coil to beta-strand transition that is independent of the peptide concentration and thus is not linked to oligomerization. Consequently, this transition may occur within the Abeta(1-40) monomer or dimer. Incubation at 37 degrees C leads to slow reversible concentration-dependent beta-sheet accumulation; heating to 85 degrees C induces further beta-sheet folding and oligomerization. Our results demonstrate the importance of temperature and thermal history for the conformation of Abeta.