A salt-induced kinetic intermediate is on a new parallel pathway of lysozyme folding.

Lysozyme folds through two competing pathways. A fast pathway leads directly from a collapsed state to the native protein, whereas folding on a slow pathway proceeds through a partially folded intermediate (I(1)). At NaCl concentrations above 100 mM, a second transient intermediate (I(2)) is induced as judged by the appearance of an additional apparent rate constant in the refolding kinetics. Monitoring the time course of native molecules and of both intermediates shows that the NaCl-induced state (I(2)) is located on neither of the two folding pathways observed at low-salt concentrations. These results suggest that I(2) is a metastable high-energy intermediate at low-ionic strength and is located on a third folding pathway. The folding landscape of lysozyme seems to be complex with several high-energy intermediates located on parallel folding routes. However, the experiments show no evidence for partially folded states on the fast direct pathway.     

Equilibrium and transient intermediates in folding of human macrophage migration inhibitory factor.

Acid, guanidinium-Cl and urea denaturations of recombinant human macrophage migration inhibitory factor (MIF) were measured using CD and fluorimetry. The acid-induced denaturation was followed by CD at 200, 222, and 278 nm and by tryptophan fluorescence. All four probes revealed an acid-denatured state below pH 3 which resembled a typical molten globule. The pH transition is not two-state as the CD data at 222 nm deviated from all other probes. Urea and guanidinium-Cl denaturations (pH 7, 25 degrees C) both gave an apparent DeltaGU app H2O of 31 +/- 3 kJ.mol-1 when extrapolated to zero denaturant concentration. However, denaturation transitions recorded by fluorescence (at the same protein concentration) occurred at lower urea or guanidinium-Cl concentrations, consistent with an intermediate in the course of MIF denaturation. CD at 222 nm was not very sensitive to protein concentration (in 10-fold range) even though size-exclusion chromatogryphy (SEC) revealed a dimer-monomer dissociation prior to MIF unfolding. Refolding experiments were performed starting from acid, guanidinium-Cl and urea-denatured states. The kinetics were multiphasic with at least two folding intermediates. The intrinsic rate constant of the main folding phase was 5.0 +/- 0.5 s-1 (36.6 degrees C, pH 7) and its energy of activation 155 +/- 12 kJ.mol-1.     

Hidden intermediates in Mango III RNA aptamer folding revealed by pressure perturbation

Fluorescent RNA aptamers have the potential to enable routine quantitation and localization of RNA molecules and serve as models for understanding biologically active aptamers. In recent years, several fluorescent aptamers have been selected and modified to improve their properties, revealing that small changes to the RNA or the ligands can modify significantly their fluorescent properties. Although structural biology approaches have revealed the bound, ground state of several fluorescent aptamers, characterization of low-abundance, excited states in these systems is crucial to understanding their folding pathways. Here we use pressure as an alternative variable to probe the suboptimal states of the Mango III aptamer with both fluorescence and NMR spectroscopy approaches. At moderate KCl concentrations, increasing pressure disrupted the G-quadruplex structure of the Mango III RNA and led to an intermediate with lower fluorescence. These observations indicate the existence of suboptimal RNA structural states that still bind the TO1-biotin fluorophore and moderately enhance fluorescence. At higher KCl concentration as well, the intermediate fluorescence state was populated at high pressure, but the G-quadruplex remained stable at high pressure, supporting the notion of parallel folding and/or binding pathways. These results demonstrate the usefulness of pressure for characterizing RNA folding intermediates.     

Highly divergent dihydrofolate reductases conserve complex folding mechanisms.

To test the hypothesis that protein folding mechanisms are better conserved than amino acid sequences, the mechanisms for dihydrofolate reductases (DHFR) from human (hs), Escherichia coli (ec) and Lactobacillus casei (lc) were elucidated and compared using intrinsic Trp fluorescence and fluorescence-detected 8-anilino-1-naphthalenesulfonate (ANS) binding. The development of the native state was monitored using either methotrexate (absorbance at 380 nm) or NADPH (extrinsic fluorescence) binding. All three homologs displayed complex unfolding and refolding kinetic mechanisms that involved partially folded states and multiple energy barriers. Although the pairwise sequence identities are less than 30 %, folding to the native state occurs via parallel folding channels and involves two types of on-pathway kinetic intermediates for all three homologs. The first ensemble of kinetic intermediates, detected within a few milliseconds, has significant secondary structure and exposed hydrophobic cores. The second ensemble is obligatory and has native-like side-chain packing in a hydrophobic core; however, these intermediates are unable to bind active-site ligands. The formation of the ensemble of native states occurs via three channels for hsDHFR, and four channels for lcDHFR and ecDHFR. The binding of active-site ligands (methotrexate and NADPH) accompanies the rate-limiting formation of the native ensemble. The conservation of the fast, intermediate and slow-folding events for this complex alpha/beta motif provides convincing evidence for the hypothesis that evolutionarily related proteins achieve the same fold via similar pathways.     

Parallel pathways in cytochrome c(551) folding

The folding of cytochrome c(551) from Pseudomonas aeruginosa was previously thought to follow a simple sequential mechanism, consistent with the lack of histidine residues, other than the native His16 heme ligand, that can give rise to mis-coordinated species. However, further kinetic analysis reveals complexities indicative of a folding mechanism involving parallel pathways. Double-jump interrupted refolding experiments at low pH indicate that approximately 50% of the unfolded cytochrome c(551) population can reach the native state via a fast (10 ms) folding track, while the rest follows a slower folding path with populated intermediates. Stopped-flow experiments using absorbance at 695 nm to monitor refolding confirm the presence of a rapidly folding species containing the native methionine-iron bond while measurements on carboxymethylated cytochrome c(551) (which lacks the Met-Fe coordination bond) indicate that methionine ligation occurs late during folding along the fast folding track, which appears to be dominant at physiological pH. Continuous-flow measurements of tryptophan-heme energy transfer, using a capillary mixer with a dead time of about 60 micros, show evidence for a rapid chain collapse within 100 micros preceding the rate-limiting folding phase on the milliseconds time scale. A third process with a time constant in the 10-50 ms time range is consistent with a minor population of molecules folding along a parallel channel, as confirmed by quantitative kinetic modeling. These findings indicate the presence of two or more slowly inter-converting ensembles of denatured states that give rise to pH-dependent partitioning among fast and slow-folding pathways.     

Combined optical and photoelectric study of the photocycle of 13-cis bacteriorhodopsin.

The photocycle of the 13-cis retinal containing bacteriorhodopsin was studied by three different techniques. The optical multichannel analyzer monitored the spectral changes during the photocycle and gave information about the number and the spectrum of the intermediates. The absorption kinetic measurements provided the possibility of following the absorbance changes at several characteristic wavelengths. The electric signal provided information about the charge motions during the photocycle. The results reveal the existence of two intermediates in the 13-cis photocycle, one with a short lifetime having an average of 1.7 microseconds and an absorption maximum at 620 nm. The other, a long-living intermediate, has a lifetime of about 50 ms and an absorption maximum around 585 nm. The data analysis suggests that these intermediates are in two parallel branches of the photocycle, and branching from the intermediate with the shorter lifetime might be responsible for the light-adaptation process.     

GroEL Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.     

Probing the folding intermediate of Rd-apocyt b562 by protein engineering and infrared T-jump

Small proteins often fold in an apparent two-state manner with the absence of detectable early-folding intermediates. Recently, using native-state hydrogen exchange, intermediates that exist after the rate-limiting transition state have been identified for several proteins. However, little is known about the folding kinetics from these post-transition intermediates to their corresponding native states. Herein, we have used protein engineering and a laser-induced temperature-jump (T-jump) technique to investigate this issue and have applied it to Rd-apocyt b(562) , a four-helix bundle protein. Previously, it has been shown that Rd-apocyt b(562) folds via an on-pathway hidden intermediate, which has only the N-terminal helix unfolded. In the present study, a double mutation (V16G/I17A) in the N-terminal helix of Rd-apocyt b(562) was made to further increase the relative population of this intermediate state at high temperature by selectively destabilizing the native state. In the circular dichroism thermal melting experiment, this mutant showed apparent two-state folding behavior. However, in the T-jump experiment, two kinetic phases were observed. Therefore, these results are in agreement with the idea that a folding intermediate is populated on the folding pathway of Rd-apocyt b(562) . Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 microsec)(-1) at 65 degrees C.     

Spectral evidence for the existence of a second cytochrome o in whole cells of Vitreoscilla

Cytochrome o, a protoheme IX pigment, has been proposed as the terminal oxidase of the filamentous bacterium, Vitreoscilla. Aerobic and anaerobic photolysis of CO-liganded whole cells demonstrated the presence of a second CO-reactive pigment, cytochrome o'. At temperatures lower than -100 degrees C, anaerobic photolysis dissociated only about 25% of the total CO-liganded components to reveal the unliganded cytochrome o'. At these temperatures, the photolysis of cytochrome o could not be demonstrated. At warmer temperatures, recombination of CO with the reduced cytochrome o' occurred with an apparent energy of activation of 5.8 kcal/mol. Aerobic photolysis of whole cells demonstrated two oxygen-bound intermediates. At temperatures lower than -95 degrees C, a spectrally distinct compound with absorption maxima at 428, 534, and 564 nm appeared (form I'); the apparent second order rate constant (k+1) for the formation of this intermediate was found to be 9.1 M-1 s-1, the reverse rate (k-1) was 9.9 X 10(-5) s-1, and the equilibrium constant (Kd) was 1.1 X 10(-5) M. This oxygen intermediate of cytochrome o' is spectrally and kinetically similar to the oxygen intermediate of cytochrome o seen in Escherichia coli. At temperatures warmer than -90 degrees C, photolysis of aerobic samples resulted in the immediate formation of a second oxygen-bound intermediate (form I) with absorption maxima at 422, 534, and 564 nm. This second intermediate results from the binding of oxygen to the cytochrome o (oxygenated cytochrome o). These data support the proposal that whole cells of Vitreoscilla contain two alternative pathways of electron transport, one terminating with cytochrome o and the other with cytochrome o'.     

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.     

Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

When a protein exhibits complex kinetics of refolding, we often ascribe the complexity to slow isomerization events in the denatured protein, such as cis/trans isomerization of peptidyl prolyl bonds. Does the complex folding kinetics arise only from this well-known reason? Here, we have investigated the refolding of a proline-free variant of staphylococcal nuclease by stopped-flow, double-jump techniques, to examine the folding reactions without the slow prolyl isomerizations. As a result, the protein folds into the native state along at least two accessible parallel pathways, starting from a macroscopically single denatured-state ensemble. The presence of intermediates on the individual folding pathways has revealed the existence of multiple parallel pathways, and is characterized by multi-exponential folding kinetics with a lag phase. Therefore, a "single" amino acid sequence can fold along the multiple parallel pathways. This observation in staphylococcal nuclease suggests that the multiple folding may be more general than we have expected, because the multiple parallel-pathway folding cannot be excluded from proteins that show simpler kinetics.     

Kinetics of an individual transmembrane helix during bacteriorhodopsin folding

The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.     

The folding of alpha-1-proteinase inhibitor: kinetic vs equilibrium control

Previous folding studies of alpha-1-proteinase inhibitor (alpha1-PI), which regulates the activity of the serine protease human neutrophil elastase, show an intermediate state at approximately 1.5 M guanidine-HCl (Gu). For the normal form of alpha1-PI, we demonstrate the reversible formation of the same stable distribution of monomeric and polymeric intermediates after approximately 1 h in 1.5 M Gu at approximately 23 degrees C from fully folded or fully unfolded alpha1-PI at similar final total concentrations and show that the stable distribution of monomeric and polymeric intermediates conforms with the law of mass action. We attribute these observations to an apparent equilibrium among intermediates. Our CD data are compatible with the intermediates having slightly relaxed structures relative to that of fully folded alpha1-PI and, thus, with the polymeric intermediates having a loop-sheet structure. Furthermore, we observe that the rates of folding (fast and slow terms) from the intermediate state are the same as those from the fully unfolded state, thereby supporting the contention that this intermediate state is on the folding pathway. We attribute the tendency of the Z mutant protein to polymerize/aggregate to an increased rate of the monomeric intermediate to form the apparent equilibrium distribution of intermediate species relative to its rate of folding to give intact alpha1-PI.     

Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.     

Characterization of Denatured States and Reversible Unfolding of Sensory Rhodopsin II

Our understanding on the folding of membrane proteins lags behind that of soluble proteins due to challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding pathways, we have systematically investigated the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 Χ_SDS), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal-binding pocket is disrupted, with transmembrane residues becoming more solvent exposed. Folding of pSRII from an SDS-denatured state harboring a covalently bound retinal chromophore shows deviations from an apparent two-state behavior. SDS denaturation to form the sensory opsin apo-protein is reversible. We report pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin.     

An on-pathway intermediate in the folding of a PDZ domain

The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.     

Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics

The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.     

Multiple folding pathways of the SH3 domain

Experimental observations suggest that proteins follow different folding pathways under different environmental conditions. We perform molecular dynamics simulations of a model of the c-Crk SH3 domain over a broad range of temperatures, and identify distinct pathways in the folding transition. We determine the kinetic partition temperature-the temperature for which the c-Crk SH3 domain undergoes a rapid folding transition with minimal kinetic barriers-and observe that below this temperature the model protein may undergo a folding transition by multiple folding pathways via only one or two intermediates. Our findings suggest the hypothesis that the SH3 domain, a protein fold for which only two-state folding kinetics was observed in previous experiments, may exhibit intermediate states under conditions that strongly stabilize the native state.