In vitro folding and oligomerization of a membrane protein. Transition of bacterial porin from random coil to native conformation.

Porin, a channel-forming protein spanning bacterial outer membranes, was denatured in 6 M guanidinium hydrochloride or, alternatively, in sodium dodecyl sulfate at 95 degrees C. Circular dichroism spectra revealed that this protein, which in its native state consist of beta-pleated sheets as the sole detectable secondary structure, is transformed into random coil configuration in the chaotropic agent, or into alpha-helical structure in the detergent. From either state, the mature protein refolds in presence of amphiphilic molecules, attaining full structural and functional competence. As structural criteria, the native trimeric state was assayed by analytical ultracentrifugation, gel electrophoresis in sodium dodecyl sulfate, protease resistance, and circular dichroism spectroscopy. Channel formation in planar lipid bilayers reveals that the refolded protein is also functionally competent. It is concluded that the information required for the complete folding of porin is contained within the primary sequence of the mature polypeptide. The study of rapid refolding clearly reveals that this process occurs in the time range of seconds and that preexisting bilayers are not a prerequisite.     

Optimizing protein folding to the native state in bacteria.

A correctly folded protein is usually both active and soluble. This review focuses on novel ways to improve the folding of recombinant proteins during production in bacteria and includes a few tips for refolding proteins. Major results in correlating protein primary structure with proper folding and stability, and the production of viral antigens and antibodies in bacteria are also discussed.     

Kinetic traps in the folding of beta alpha-repeat proteins: CheY initially misfolds before accessing the native conformation

The βα-repeat class of proteins, represented by the (βα)₈ barrel and the α/β/α sandwich, are among the most common structural platforms in biology. Previous studies on the folding mechanisms of these motifs have revealed or suggested that the initial event involves the submillisecond formation of a kinetically trapped species that must at least partially unfold before productive folding to the respective native conformation can occur. To test the generality of these observations, CheY, a bacterial response regulator, was subjected to an extensive analysis of its folding reactions. Although earlier studies had proposed the formation of an off-pathway intermediate, the data available were not sufficient to rule out an alternative on-pathway mechanism. A global analysis of single- and double-jump kinetic data, combined with equilibrium unfolding data, was used to show that CheY folds and unfolds through two parallel channels defined by the state of isomerization of a prolyl peptide bond in the active site. Each channel involves a stable, highly structured folding intermediate whose kinetic properties are better described as the properties of an off-pathway species. Both intermediates subsequently flow through the unfolded state ensemble and adopt the native cis-prolyl isomer prior to forming the native state. Initial collapse to off-pathway folding intermediates is a common feature of the folding mechanisms of βα-repeat proteins, perhaps reflecting the favored partitioning to locally determined substructures that cannot directly access the native conformation. Productive folding requires the dissipation of these prematurely folded substructures as a prelude to forming the larger-scale transition state that leads to the native conformation. Results from Gō-modeling studies in the accompanying paper elaborate on the topological frustration in the folding free-energy landscape of CheY.     

Protein folding in vitro.

It is becoming increasingly evident that intermediates observed in protein folding in vitro may be closely related to conformational states that are important in various intracellular processes. This review focuses on recent advances in in vitro protein-folding studies with particular reference to the molten globule state, which is purported to be a common and distinct intermediate of protein folding.     

Productive folding to the native state by a group II intron ribozyme.

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.     

Using motion planning to map protein folding landscapes and analyze folding kinetics of known native structures.

We investigate a novel approach for studying the kinetics of protein folding. Our framework has evolved from robotics motion planning techniques called probabilistic roadmap methods (PRMs) that have been applied in many diverse fields with great success. In our previous work, we presented our PRM-based technique and obtained encouraging results studying protein folding pathways for several small proteins. In this paper, we describe how our motion planning framework can be used to study protein folding kinetics. In particular, we present a refined version of our PRM-based framework and describe how it can be used to produce potential energy landscapes, free energy landscapes, and many folding pathways all from a single roadmap which is computed in a few hours on a desktop PC. Results are presented for 14 proteins. Our ability to produce large sets of unrelated folding pathways may potentially provide crucial insight into some aspects of folding kinetics, such as proteins that exhibit both two-state and three-state kinetics that are not captured by other theoretical techniques.     

A basic residue at position 36p of the propeptide is not essential for the correct folding and subsequent autocatalytic activation of prochymosin.

Position 36p in the propeptides of gastric aspartic proteinases is generally occupied by lysine or arginine. This has led to the conclusion that a basic residue at this position, which interacts with the active-site aspartates, is essential for folding and activation of the zymogen. Lamb prochymosin has been shown by cDNA cloning to possess glutamic acid at 36p. To investigate the effect of this natural mutation which appears to contradict the proposed role of this residue, calf and lamb prochymosins and their two reciprocal mutants, K36pE and E36pK, respectively, were expressed in Escherichia coli, refolded in vitro, and autoactivated at pH 2 and 4.7. All four zymogens could be activated to active chymosin and, at both pH values, the two proteins with Glu36p showed higher activation rates than the two Lys36p forms. Glu36p was also demonstrated in natural prochymosin isolated from the fourth stomach of lamb, as well as being encoded in the genomes of sheep, goat and mouflon, which belong to the subfamily Caprinae. A conserved basic residue at position 36p of prochymosin is thus not obligatory for its folding or autocatalytic activation. The apparently contradictory results for porcine pepsinogen A [Richter, C., Tanaka, T., Koseki, T. & Yada, R.Y. (1999) Eur. J. Biochem. 261, 746-752] can be reconciled with those for prochymosin. Lys/Arg36p is involved in stabilizing the propeptide-enzyme interaction, along with residues nearer the N-terminus of the propeptide, the sequence of which varies between species. The relative contribution of residue 36p to stability differs between pepsinogen and prochymosin, being larger in the former.     

Protein folding. Effect of packing density on chain conformation

Recent lattice polymer simulations by Chan & Dill suggest that compactness may be a significant driving force in the formation of secondary structure. We have addressed the robustness of this conclusion for non-lattice polymers using a rotational isomeric model of proteins. Boundary conditions are used to enforce compactness and excluded volume effects are explicitly incorporated. As in the cubic lattice studies, compactness is seen to influence secondary structure content. This effect is modest for densities comparable to native proteins but dramatic for chains that are approximately 30% more dense than native proteins. alpha-Helical structure is common but beta-sheet structure is rare. It appears that lattices impart to compact chains an organizational bias that favors beta-sheet structure. The strengths and weakness of various simplified representations of polypeptide chains are also discussed.     

Regeneration of native ovalbumin conformation disrupted by citraconylation.

Ovalbumin was reacted with a 960-fold molar excess of citraconic anhydride, and 91% of the epsilon-amino groups, representing 18 of the 20 lysine residues, were citraconylated. As detected by fluorescence and far-ultraviolet circular dichroic (CD) measurements, the modified protein displayed significant disruption of the native conformation. Treatment at pH 2.2 for 5 h resulted in the hydrolysis of 10 of the 18 citraconyl groups, but when subjected to the acid conditions for 12 h, all 18 modifying groups were removed. Electrophoretically, the 5-h and the 12-h acid-treated proteins were homogeneous and showed decreasing anodic mobility at pH 8.3; indeed, the anodic mobility of the 12-h acid-treated protein was identical to that of the native protein. Similarly, the 12-h acid-treated protein possessed conformational properties almost indistinguishable from the native protein. These properties included similar emission fluorescence spectra and far-ultraviolet CD spectra, similar resistance to undergoing helix-to-coil transition at pH 12.2, and identical urea unfolding curves, and thus identical urea transition mid-point of about 8.0 M. These observations indicate that the protein with all the epsilon-amino groups regenerated by acid treatment has the same conformational stability as the native ovalbumin.     

Refolding processes of cytochrome P450cam from ferric and ferrous acid forms to the native conformation. Formations of folding intermediates with non-native heme coordination state

Changes in heme coordination state and protein conformation of cytochrome P450(cam) (P450(cam)), a b-type heme protein, were investigated by employing pH jump experiments coupled with time-resolved optical absorption, fluorescence, circular dichroism, and resonance Raman techniques. We found a partially unfolded form (acid form) of ferric P450(cam) at pH 2.5, in which a Cys(-)-heme coordination bond in the native conformation was ruptured. When the pH was raised to pH 7.5, the acid form refolded to the native conformation through a distinctive intermediate. Formations of similar acid and intermediate forms were also observed for ferrous P450(cam). Both the ferric and ferrous forms of the intermediate were found to have an unidentified axial ligand of the heme at the 6th coordination sphere, which is vacant in the high spin ferric and ferrous forms at the native conformation. For the ferrous form, it was also indicated that the 5th axial ligand is different from the native cysteinate. The folding intermediates identified in this study demonstrate occurrences of non-native coordination state of heme during the refolding processes of the large b-type heme protein, being akin to the well known folding intermediates of cytochromes c, in which c-type heme is covalently attached to a smaller protein.     

Evidence contrary to the protein error hypothesis for in vitro senescence.

A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucose-6-phosphate dehydrogenase (G-6-PD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 +/- 1%, S.E.), in comparison with that in 19 old cultures (4.9 +/- 1%, S.E.). Old cultures had ceased active cell division (49-60 doublings); DNA replication, measured by [3H]thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5-22 doublings) had a [3H]thymidine labeling index of 75-85%. Titration of G-6-PD activity in extracts of young and old cells with neutralizing antibody directes specifically against G-6-PD failed to detect an increment of enzymatically defective G-6-PD in old cells. The thermostability studies were capable of detecting altered G-6-PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G-6-PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G-6-PD from the thermolabile mutant and G-6-PD inactivated with N-ethylameimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.     

The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation.

The hammerhead ribozyme undergoes an ion-dependent folding process into the active conformation. We find that the folding can be blocked at specific stages by changes of sequence or functionality within the core. In the the absence of added metal ions, the global structure of the hammerhead is extended, with a large angle subtended between stems I and II. No core sequence changes appear to alter this geometry, consistent with an unstructured core under these conditions. Upon addition of low concentrations of magnesium ions, the hammerhead folds by an association of stems II and III, to include a large angle between them. This stage is inhibited or altered by mutations within the oligopurine sequence lying between stems II and III, and folding is completely prevented by an A14G mutation. Further increase in magnesium ion concentration brings about a second stage of folding in the natural sequence hammerhead, involving a reorientation of stem I, which rotates around into the same direction of stem II. Because this transition occurs over the same range of magnesium ion concentration over which the hammerhead ribozyme becomes active, it is likely that the final conformation is most closely related to the active form of the structure. Magnesium ion-dependent folding into this conformation is prevented by changes at G5, notably removal of the 2'-hydroxyl group and replacement of the base by cytidine. The ability to dissect the folding process by means of sequence changes suggests that two separate ion-dependent stages are involved in the folding of the hammerhead ribozyme into the active conformation.     

Properties of the collagen type XVII ectodomain. Evidence for n- to c-terminal triple helix folding

Collagen XVII is a transmembrane component of hemidesmosomal cells with important functions in epithelial-basement membrane interactions. Here we report on properties of the extracellular ectodomain of collagen XVII, which harbors multiple collagenous stretches. We have recombinantly produced subdomains of the collagen XVII ectodomain in a mammalian expression system. rColXVII-A spans the entire ectodomain from deltaNC16a to NC1, rColXVII-B is similar but lacks the NC1 domain, a small N-terminal polypeptide rColXVII-C encompasses domains deltaNC16a to C15, and a small C-terminal polypeptide rColXVII-D comprises domains NC6 to NC1. Amino acid analysis of rColXVII-A and -C demonstrated prolyl and lysyl hydroxylation with ratios for hydroxyproline/proline of 0.4 and for hydroxylysine/lysine of 0.5. A small proportion of the hydroxylysyl residues in rColXVII-C ( approximately 3.3%) was glycosylated. Limited pepsin and trypsin degradation assays and analyses of circular dichroism spectra clearly demonstrated a triple-helical conformation for rColXVII-A, -B, and -C, whereas the C-terminal rColXVII-D did not adopt a triple-helical fold. These results were further substantiated by electron microscope analyses, which revealed extended molecules for rColXVII-A and -C, whereas rColXVII-D appeared globular. Thermal denaturation experiments revealed melting temperatures of 41 degrees C (rColXVII-A), 39 degrees C (rColXVII-B), and 35 degrees C (rColXVII-C). In summary, our data suggest that triple helix formation in the ectodomain of ColXVII occurs with an N- to C-terminal directionality.     

Effects of low concentrations of guanidine . HCl on the reconstitution of lactic dehydrogenase from pig muscle in vitro. Evidence for guanidine binding to the native enzyme

The presence of low concentrations of guanidine . HCl has a pronounced effect on the overall rate of reactivation of lactic dehydrogenase from pig muscles after preceding dissociation and deactivation in various denaturants. The obseverd attenuation is a function of the amount of guanidine . HCl present during reconstitution. At a given guanidine concentration in the reactivation buffer the yield, but not the rate of reactivation, is influenced by the extent of denaturation caused initially in the process of deactivation and dissociation. As a possible explanation for the influence of guanidine . HCl on the kinetics of reconstitution, binding of the ligand to intermediates of folding and association is considered. This hypothesis is corroborated by the observation that guanidine . HCl in the relevant concentration range does bind to native lactic dehydrogenase without inactivating the enzyme or disrupting its quaternary structure. A kinetic model comprising guanidine binding to both the native enzyme and structured intermediates is proposed to describe the observed effects of guanidine . HCl on the rate of reactivation. In addition, the dissociation constants for guanidine binding to intermediates of reconstitution and to native lactic dehydrogenase are estimated.     

Comparing the energy landscapes for native folding and aggregation of PrP

Protein sequences are evolved to encode generally one folded structure, out of a nearly infinite array of possible folds. Underlying this code is a funneled free energy landscape that guides folding to the native conformation. Protein misfolding and aggregation are also a manifestation of free-energy landscapes. The detailed mechanisms of these processes are poorly understood, but often involve rare, transient species and a variety of different pathways. The inherent complexity of misfolding has hampered efforts to measure aggregation pathways and the underlying energy landscape, especially using traditional methods where ensemble averaging obscures important rare and transient events. We recently studied the misfolding and aggregation of prion protein by examining 2 monomers tethered in close proximity as a dimer, showing how the steps leading to the formation of a stable aggregated state can be resolved in the single-molecule limit and the underlying energy landscape thereby reconstructed. This approach allows a more quantitative comparison of native folding versus misfolding, including fundamental differences in the dynamics for misfolding. By identifying key steps and interactions leading to misfolding, it should help to identify potential drug targets. Here we describe the importance of characterizing free-energy landscapes for aggregation and the challenges involved in doing so, and we discuss how single-molecule studies can help test proposed structural models for PrP aggregates.     

Monitoring protein conformation along the pathway of chaperonin-assisted folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.