Cooperative assembly of a nativelike ubiquitin structure through peptide fragment complexation: energetics of peptide association and folding

Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.     

Limited conformational space for early-stage protein folding simulation

MOTIVATION: The problem of early-stage protein folding is critical for protein structure prediction. The model presented introduces a common definition of protein structures which may be treated as the possible in silico early-stage form of the polypeptide chain. Limitation of the conformational space to the ellipse path on the Ramachandran map was tested as a possible sub-space to represent the early-stage structure for simulation of protein folding. The proposed conformational sub-space was developed on the basis of the backbone conformation, with side-chain interactions excluded. RESULTS: The ellipse-path-limited conformation of BPTI was created using the criterion of shortest distance between Phi, Psi angles in native form of protein and the Phi, Psi angles belonging to the ellipse. No knots were observed in the structure created according to ellipse-path conformational sub-space. The energy minimization procedure applied to ellipse-path derived conformation directed structural changes toward the native form of the protein with SS-bonds system introduced to the procedure. AVAILABILITY: Program 'Ellipse' to create the ellipse-path derived structure available on request: myroterm@cyf-kr.edu.pl     

The folding funnel landscape for the peptide Met-enkephalin

We study the free energy landscape of the small peptide Met-enkephalin. Our data were obtained from a generalized-ensemble Monte Carlo simulation taking the interactions among all atoms into account. We show that the free energy landscape resembles that of a funnel, indicating that this peptide is a good folder. Our work demonstrates that the energy landscape picture and folding concept, developed in the context of simplified protein models, can also be used to describe the folding in more realistic models.     

Kinetics and energetics of the translocation of maltose binding protein folding mutants

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB.     

An in vitro peptide folding model suggests the presence of the molten globule state during nascent peptide folding

Although molten globules have been widely accepted as a general intermediate in protein folding, there is no clear evidence to show their presence during nascent peptide folding. This paper concentrates on whether the molten globule state occurs, and if it does, when does it form during nascent peptide folding, by comparing the changes in conformation during peptide chain extension of staphylococcal nuclease R. The results show that a large N-terminal fragment of staphylococcal nuclease, SNR121, which already contains more than 80% amino acid sequence of the nuclease, is found to fulfill all the criteria for the molten globule state, suggesting that the molten globule should occur at a later stage of peptide elongation. At this stage the hydrophobic collapse of the polypeptide chain occurs driven by the hydrophobic force, which leads to the formation of a solvent-accessible non-polar core, characterized by the high ANS-binding fluorescence. The nascent peptide folding of the nuclease is a hierarchical process that at the very least includes the following steps: secondary structure accumulation, pre-molten globule state, molten globule state, post-molten globule state and finally the native state. Constant conformation adjustment is necessary for correct folding and active expression of the protein.     

Cooperative folding of tau peptide by coordination of group IIB metal cations during heparin-induced aggregation

The group IIB elements, especially Cd(II) and Hg(II), are increasingly considered as potential environmental neurotoxins. This study demonstrates that the Alzheimer’s tau fragment R2, corresponding to the second repeat of the microtubule-binding domain, can bind to Zn(II), Cd(II) and Hg(II). Isothermal titration calorimetry experiments suggest that the most likely coordination site is the thiol group of Cys291, and this is further confirmed by a control experiment using a C291A mutant peptide. Circular dichroism spectrum reveals that the coordination of group IIB cations, especially Hg(II), can induce pronounced conformational conversions in natively unfolded R2, from random coil to other ordered structures. ThS fluorescence assays and electron microscopy indicate that the group IIB cations promote heparin-induced aggregation of R2, giving relatively small R2 filaments. The efficiency in promoting aggregation, as well as inducing conformational conversion, varies strongly with the cation’s polarizability. Based on these results, a model is proposed in which the cooperative folding of R2 through cross-bridging of group IIB cations is suggested to be a key factor in promoting aggregation, in addition to the effective neutralization of coulombic charge–charge repulsion by heparin, the poly-anion inducer. Our results provide clues to understanding the potential pathogenic role of group IIB metals in the development of neurofibrillary tangles, a typical hallmark of Alzheimer’s disease.     

Redesigning the folding energetics of a model three-helix bundle protein by site-directed mutagenesis

Because of their limited size and complexity, de novo designed proteins are particularly useful for the detailed investigation of folding thermodynamics and mechanisms. Here, we describe how subtle changes in the hydrophobic core of a model three-helix bundle protein (GM-0) alter its folding energetics. To explore the folding tolerance of GM-0 toward amino acid sequence variability, two mutant proteins (GM-1 and GM-2) were generated. In the mutants, cavities were created in the hydrophobic core of the protein by either singly (GM-1; L35A variant) or doubly (GM-2; L35A/I39A variant) replacing large hydrophobic side chains by smaller Ala residues. The folding of GM-0 is characterized by two partially folded intermediate states exhibiting characteristics of molten globules, as evidenced by pressure-unfolding and pressure-assisted cold denaturation experiments. In contrast, the folding energetics of both mutants, GM-1 and GM-2, exhibit only one folding intermediate. Our results support the view that simple but biologically important folding motifs such as the three-helix bundle can reveal complex folding plasticity, and they point to the role of hydrophobic packing as a determinant of the overall stability and folding thermodynamic of the helix bundle.     

Glycopeptide probes for understanding peptide specificity of the folding sensor enzyme UGGT

A systematic series of chitobiose-modified pentapeptides with sequence variations of hydrophobic leucine and hydrophilic serine were synthesized. The resulting glycopeptides were used as molecular probes to elucidate aglycon peptide specificity of the glycoprotein-folding sensor enzyme UGGT. Inhibitory experiments with a synthetic fluorescent glyco-substrate and the glycopeptides revealed that UGGT prefers a serine residue directly linked to C-terminal of the N-glycosylation site in its substrate recognition.     

Calorimetric determination of the energetics of the molten globule intermediate in protein folding: apo-alpha-lactalbumin

High-sensitivity differential scanning calorimetry has been used to characterize the energetics of the molten globule state of apo-alpha-lactalbumin. This characterization has been possible by performing temperature scans at different guanidine hydrochloride (GuHCl) concentrations in order to experimentally define the temperature-GuHCl stability surface of the protein. Multidimensional analysis of the heat capacity surface has allowed simultaneous resolution of the energetics of the unfolded and molten globule states. These experiments indicate that the intrinsic enthalpy difference (i.e., excluding additional contributions such as those arising from differential GuHCl binding) between the unfolded and native states is 31.8 kcal/mol at 25 degrees C whereas that of the molten globule and native states is only 7.7 kcal/mol. At the same temperature, the entropy changes are 99.2 and 23.7 cal/K.mol and the heat capacity changes are 1821 and 326 cal/K.mol, respectively. Analysis of the thermodynamic data indicates that in passing from the native to the molten globule state only approximately 19% of the hydrogen bonds are broken. In addition, the magnitude of delta Cp for the molten globule suggests that water does not largely penetrate into the interior of the molten globule, implying that significant hydrophobic interactions are still present in this state. These parameters provide precise energetic constraints to the allowed structural conformations of the molten globule.     

A chemical method for investigating disulfide-coupled peptide and protein folding

Investigations of protein folding have largely involved studies using disulfide-containing proteins, as disulfide-coupled folding of proteins permits the folding intermediates to be trapped and their conformations determined. Over the last decade, a combination of new biotechnical and chemical methodology has resulted in a remarkable acceleration in our understanding of the mechanism of disulfide-coupled protein folding. In particular, expressed protein ligation, a combination of native chemical ligation and an intein-based approach, permits specifically labeled proteins to be easily produced for studies of protein folding using biophysical methods, such as NMR spectroscopy and X-ray crystallography. A method for regio-selective formation of disulfide bonds using chemical procedures has also been established. This strategy is particularly relevant for the study of disulfide-coupled protein folding, and provides us not only with the native conformation, but also the kinetically trapped topological isomer with native disulfide bonds. Here we review recent developments and applications of biotechnical and chemical methods to investigations of disulfide-coupled peptide and protein folding. Chemical additives designed to accelerate correct protein folding and to avoid non-specific aggregation are also discussed.     

An alternative route for the folding of large RNAs: apparent two-state folding by a group II intron ribozyme

Despite a growing literature on the folding of RNA, our understanding of tertiary folding in large RNAs derives from studies on a small set of molecular examples, with primary focus on group I introns and RNase P RNA. To broaden the scope of RNA folding models and to better understand group II intron function, we have examined the tertiary folding of a ribozyme (D135) that is derived from the self-splicing ai5gamma intron from yeast mitochondria. The D135 ribozyme folds homogeneously and cooperatively into a compact, well-defined tertiary structure that includes all regions critical for active-site organization and substrate recognition. When D135 was treated with increasing concentrations of Mg(2+) and then subjected to hydroxyl radical footprinting, similar Mg(2+) dependencies were seen for internalization of all regions of the molecule, suggesting a highly cooperative folding behavior. In this work, we show that global folding and compaction of the molecule have the same magnesium dependence as the local folding previously observed. Furthermore, urea denaturation studies indicate highly cooperative unfolding of the ribozyme that is governed by thermodynamic parameters similar to those for forward folding. In fact, D135 folds homogeneously and cooperatively from the unfolded state to its native, active structure, thereby demonstrating functional reversibility in RNA folding. Taken together, the data are consistent with two-state folding of the D135 ribozyme, which is surprising given the size and multi-domain structure of the RNA. The findings establish that the accumulation of stable intermediates prior to formation of the native state is not a universal feature of RNA folding and that there is an alternative paradigm in which the folding landscape is relatively smooth, lacking rugged features that obstruct folding to the native state.     

NMR refinement and peptide folding using the GROMACS software

Journal of Biomolecular NMR - Nuclear magnetic resonance spectroscopy is used routinely for studying the three-dimensional structures and dynamics of proteins and nucleic acids. Structure...     

A directed essential dynamics simulation of peptide folding

We present a directed essential dynamics (DED) method for peptide and protein folding. DED is a molecular dynamics method based on the essential dynamics sampling and the principal component analysis. The main idea of DED is to use principal component analysis to determine the direction of the most active collective motion of peptides at short intervals of time (20 fs) during the folding process and then add an additional force along it to adjust the folding direction. This method can make the peptides avoid being trapped in the local minima for a long time and enhance the sampling efficiency in conformational space during the simulation. An S-peptide with 15 amino acids is used to demonstrate the DED method. The results show that DED can lead the S-peptide to fold quickly into the native state, whereas traditional molecular dynamics needs more time to do this.     

Critical role of N-terminal N-glycosylation for proper folding of the human formyl peptide receptor

The human formyl peptide receptor (FPR) is N-glycosylated and activates phagocytes via G(i)-proteins. The FPR expressed with G(i)alpha(2)beta(1)gamma(2) in Sf9 insect cells exhibits high constitutive activity as assessed by strong inhibitory effects of an inverse agonist and Na(+) on basal guanosine 5(')-O-(3-thiotriphosphate) (GTPgammaS) binding. The aim of our study was to analyze the role of N-glycosylation in FPR function. Site-directed mutagenesis of extracellular Asn residues prevented FPR glycosylation but not FPR expression in Sf9 membranes. However, in terms of high-affinity agonist binding, kinetics of GTPgammaS binding, number of G(i)-proteins activated, and constitutive activity, non-glycosylated FPR was much less active than native FPR. FPR-Asn4Gln/Asn10Gln/Asn179Gln and FPR-Asn4Gln/Asn10/Gln exhibited similar defects. Our data indicate that N-glycosylation of N-terminal Asn4 and Asn10 but not of Asn179 in the second extracellular loop is essential for proper folding and, hence, function of FPR. FPR deglycosylation by bacterial glycosidases could be a mechanism by which bacteria compromise host defense.     

Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.     

Measuring denatured state energetics: deviations from random coil behavior and implications for the folding of iso-1-cytochrome c

The changes in the free energy of the denatured state of a set of yeast iso-1-cytochrome c variants with single surface histidine residues have been measured in 3 M guanidine hydrochloride. The thermodynamics of unfolding by guanidine hydrochloride is also reported. All variants have decreased stability relative to the wild-type protein. The free energy of the denatured state was determined in 3 M guanidine hydrochloride by evaluating the strength of heme-histidine ligation through determination of the pK(a) for loss of histidine binding to the heme. The data are corrected for the presence of the N-terminal amino group which also ligates to the heme under similar solution conditions. Significant deviations from random coil behavior are observed. Relative to a variant with a single histidine at position 26, residual structure of the order of -1.0 to -2.5 kcal/mol is seen for the other variants studied. The data explain the slower folding of yeast iso-1-cytochrome c relative to the horse protein. The greater number of histidines and the greater strength of ligation are expected to slow conversion of the histidine-misligated forms to the obligatory aquo-heme intermediate during the ligand exchange phase of folding. The particularly strong association of histidine residues at positions 54 and 89 may indicate regions of the protein with strong energetic propensities to collapse against the heme during early folding events, consistent with available data in the literature on early folding events for cytochrome c.     

Prosequence-mediated disulfide coupled folding of the peptide hormones guanylin and uroguanylin

In contrast to their prohormones the mature peptide hormones guanylin and uroguanylin are not able to fold to their native disulfide connectivities upon oxidative folding. Structural properties of both peptide hormones and their precursor proteins as well as the role of their prosequences in proper disulfide coupled folding are reviewed. In addition, the structural behavior of a proguanylin mutant that closely resembles prouroguanylin has been investigated to gain further insight into structural properties of this homologous precursor protein.     

Structured pathway across the transition state for peptide folding revealed by molecular dynamics simulations

Small globular proteins and peptides commonly exhibit two-state folding kinetics in which the rate limiting step of folding is the surmounting of a single free energy barrier at the transition state (TS) separating the folded and the unfolded states. An intriguing question is whether the polypeptide chain reaches, and leaves, the TS by completely random fluctuations, or whether there is a directed, stepwise process. Here, the folding TS of a 15-residue β-hairpin peptide, Peptide 1, is characterized using independent 2.5 μs-long unbiased atomistic molecular dynamics (MD) simulations (a total of 15 μs). The trajectories were started from fully unfolded structures. Multiple (spontaneous) folding events to the NMR-derived conformation are observed, allowing both structural and dynamical characterization of the folding TS. A common loop-like topology is observed in all the TS structures with native end-to-end and turn contacts, while the central segments of the strands are not in contact. Non-native sidechain contacts are present in the TS between the only tryptophan (W11) and the turn region (P7-G9). Prior to the TS the turn is found to be already locked by the W11 sidechain, while the ends are apart. Once the ends have also come into contact, the TS is reached. Finally, along the reactive folding paths the cooperative loss of the W11 non-native contacts and the formation of the central inter-strand native contacts lead to the peptide rapidly proceeding from the TS to the native state. The present results indicate a directed stepwise process to folding the peptide.