Studies of the collagen-like peptide (Pro-Pro-Gly)(10) confirm that the shape and position of the type I collagen denaturation endotherm is governed by the rate of helix unfolding

The kinetics of unfolding of a collagen-like peptide, (Pro-Pro-Gly)(10), has been studied under isothermal conditions to gain a better understanding of the stabilization of the collagen triple helix. The formation process was third-order and relatively insensitive to temperature at concentrations of 1 mg/ml and below, while the unfolding process was first-order and highly temperature-dependent. The helix-coil transition was studied over a range of scanning rates and polymer concentrations, using differential scanning calorimetry and the observations were compared with solutions of an approximate differential equation governing the process. At high concentrations (24 mg/ml) and very low scanning rates (0.025 degrees C min(-1)), the helicity, F, approached a quasistatic state in which it reached its equilibrium value at all temperatures. Under these conditions, the temperature at which the endotherm peaked, T(max), increased with chain concentration but was independent of scanning rate, while (dF/dT)(max) was dependent on the van't Hoff enthalpy and on the order of the formation process. On scanning from a low to a high temperature (up-scanning) at low concentrations (0.25-1.0 mg/ml) and higher scanning rates (0.1 degrees C min(-1) and above), the peak in dF/dT was taller and narrower than for slow quasistatic scanning. T(max) increased linearly with the logarithm of the scanning rate, and was independent of concentration, while (dF/dT)(max) was governed by the temperature-dependence of the rate of unfolding. At intermediate scanning rates, two peaks in dF/dT were apparent. One peak was a nascent "quasistatic peak"; the other was a nascent "rate peak". Comparison of this peptide data with the properties of the collagen denaturation endotherm showed that the collagen denaturation endotherm was determined only by the rate of unfolding, and not by an unobserved equilibrium.     

Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or are formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown limits the rate of folding to the native state. A temperature jump (T-jump) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape.     

The folding mechanism of collagen-like model peptides explored through detailed molecular simulations

Collagen has a unique folding mechanism that begins with the formation of a triple-helical structure near its C terminus followed by propagation of this structure to the N terminus. To elucidate factors that affect the folding of collagen, we explored the folding pathway of collagen-like model peptides using detailed molecular simulations with explicit solvent. Using biased molecular dynamics we examined the latter stages of folding of a peptide model of native collagen, (Pro-Hyp-Gly)10, and a peptide that models a Gly --> Ser mutation found in several forms of osteogenesis imperfecta, (Pro-Hyp-Gly)3-Pro-Hyp-Ser-(Pro-Hyp-Gly)6. Starting from an unfolded state that contains a C-terminal nucleated trimer, (Pro-Hyp-Gly)10 folds to a structure where two of the three chains associate through water-mediated hydrogen bonds and the third is relatively separated from this dimer. Calculated free-energy profiles for folding from this intermediate to the final triple-helical structure suggest that further folding occurs at a rate of approximately one Pro-Hyp-Gly triplet per msec. In contrast, after 6 nsec of biased dynamics, the region N-terminal to the Ser residue in (Pro-Hyp-Gly)3-Pro-Hyp-Ser-(Pro-Hyp-Gly)6 folds to a structure where the three chains form close contacts near the N terminus, away from the mutation site. Further folding to an ideal triple-helical structure at the site of the mutation is unfavorable as the free energy of a triple-helical conformation at this position is more than 20 kcal/mol higher than that of a structure with unassociated chains. These data provide insights into the folding pathway of native collagen and the events underlying the formation of misfolded structures.     

Submillisecond folding of the peripheral subunit-binding domain.

Folding and unfolding rates have been measured for the peripheral subunit-binding domain, a small three-helix protein. The protein folds very fast, with rates too rapid to be measured using traditional stopped-flow techniques. Folding and unfolding rates were measured as a function of temperature using dynamic NMR lineshape analysis. At the lowest temperature at which there is sufficient broadening to measure rates, 41 degrees C, the folding rate is 16,050 s(-1). Thus, the halftime required for folding is 43 micros. At the same temperature, the unfolding rate is 2800 s(-1). Identical rates were measured using resolved resonances from Val16 in the loop and Val21 at the end of the 310-helix. Folding rates have been correlated with protein topology, and this correlation is consistent with the rapid folding of the peripheral subunit-binding domain. The results presented here show that the peripheral subunit-binding domain is the third fastest folding protein for which rates have been estimated. The folding rate is the fastest that has been directly measured and provides further support for the importance of chain topology as a major determinant of folding rates.     

Asymmetry in the triple helix of collagen-like heterotrimers confirms that external bonds stabilize collagen structure

Heating and subsequent cooling mixtures of (Pro-Pro-Gly)(10) and (Pro-Hyp-Gly)(10) peptides leads to formation of model heterotrimeric collagen helices that can be isolated by HPLC. These heterotrimeric collagen peptide helices are shown to be fundamentally unstable as denaturing then renaturing experiments result in heterotrimeric/homotrimeric mixtures.As the proportion of hydroxyproline-containing chains in the trimers increases, differential scanning calorimetry shows that the helix melting temperatures and denaturation enthalpies increasing non-linearly. Three types of Rich-Crick hydrogen bonds observed by NMR allow modelling of heterotrimeric structures based on published homotrimeric X-ray data. This revealed a small axial movement of (Pro-Hyp-Gly)(10) chains towards the C-terminal of the helix, demonstrating heterotrimeric asymmetry.     

The osmolyte trimethylamine‐N‐oxide stabilizes the Fyn SH3 domain without altering the structure of its folding transition state

Trimethylamine‐N‐oxide (TMAO) is a naturally occurring osmolyte that stabilizes proteins against denaturation. Although the impact of TMAO on the folding thermodynamics of many proteins has been well characterized, far fewer studies have investigated its effects on protein folding kinetics. In particular, no previous studies have used Φ‐value analysis to determine whether TMAO may alter the structure of the folding transition state. Here we have measured the effects on folding kinetics of 16 different amino acid substitutions distributed across the structure of the Fyn SH3 domain both in the presence and absence of TMAO. The folding and unfolding rates in TMAO, on average, improved to equivalent degrees, with a twofold increase in the protein folding rate accompanied by a twofold decrease in the unfolding rate. Importantly, TMAO caused little alteration to the Φ‐values of the mutants tested, implying that this compound minimally perturbs the folding transition state structure. Furthermore, the solvent accessibility of the transition state was not altered as reflected in an absence of a TMAO‐induced change in the denaturant β factors. Through TMAO‐induced folding studies, a β factor of 0.5 was calculated for this compound, suggesting that the protein backbone, which is the target of action of TMAO, is 50% exposed in the transition state as compared to the native state. This finding is consistent with the equivalent effects of TMAO on the folding and unfolding rates. Through thermodynamic analysis of mutants, we also discovered that the stabilizing effect of TMAO is lessened with increasing temperature.     

Kinetically robust monomeric protein from a hyperthermophile

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.     

Observation of multistate kinetics during the slow folding and unfolding of barstar.

The kinetics of the slow folding and unfolding reactions of barstar, a bacterial ribonuclease inhibitor protein, have been studied at 23(+/-1) degrees C, pH 8, by the use of tryptophan fluorescence, far-UV circular dichroism (CD), near-UV CD, and transient mixing (1)H nuclear magnetic resonance (NMR) spectroscopic measurements in the 0-4 M range of guanidine hydrochloride (GdnHCl) concentration. The denaturant dependences of the rates of folding and unfolding processes, and of the initial and final values of optical signals associated with these kinetic processes, have been determined for each of the four probes of measurement. Values determined for rates as well as amplitudes are shown to be very much probe dependent. Significant differences in the intensities and rates of appearance and disappearance of several resolved resonances in the real-time one-dimensional NMR spectra have been noted. The NMR spectra also show increasing dispersion of chemical shifts during the slow phase of refolding. The denaturant dependences of rates display characteristic folding chevrons with distinct rollovers under strongly native as well as strongly unfolding conditions. Analyses of the data and comparison of the results obtained with different probes of measurement appear to indicate the accumulation of a myriad of intermediates on parallel folding and unfolding pathways, and suggest the existence of an ensemble of transition states. The energetic stabilities of the intermediates estimated from kinetic data suggest that they are approximately half as stable as the fully folded protein. The slowness of the folding and unfolding processes (tau = 10-333 s) and values of 20.5 (+/-1.4) and 18 (+/-0.5) kcal mol(-)(1) for the activation energies of the slow refolding and unfolding reactions suggest that proline isomerization is involved in these reactions, and that the intermediates accumulate and are therefore detectable because the slow proline isomerization reaction serves as a kinetic trap during folding.     

Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

Rate-temperature relationships in lambda-repressor fragment lambda 6-85 folding

Two classes of lambda(6-85) mutants (those richer in alanine, and those richer in glycine) have very similar slopes in an Arrhenius plot of the unfolding rates but very different temperature dependencies of the folding rates. Temperature-dependent interactions (e.g., hydrophobicity) play a large role in the initial stages of folding but not in the initial stages of unfolding of lambda(6-85). Placement of the transition state in terms of its surface exposure and entropy shows that at least two reaction coordinates are required to describe folding of all mutants over the full temperature range. The unusual Arrhenius plots of the very fastest mutant provide an additional kinetic signature for downhill folding.     

Tuning lambda6-85 towards downhill folding at its melting temperature

The five-helix bundle lambda6-85* is a fast two-state folder. Several stabilized mutants have been reported to fold kinetically near-downhill or downhill. These mutants undergo a transition to two-state folding kinetics when heated. It has been suggested that this transition is caused by increased hydrophobicity at higher temperature. Here we investigate two histidine-containing mutants of lambda6-85* to see if a weaker hydrophobic core can extend the temperature range of downhill folding. The very stable lambdaHA is the fastest-folding lambda repressor to date (k(f)(-1) approximately k(obs)(-1)=2.3 micros at 44 degrees C). It folds downhill at low temperature, but transits back to two-state folding at its unfolding midpoint. lambdaHG has a weakened hydrophobic core. It is less stable than some slower folding mutants of lambda6-85*, and it has more exposed hydrophobic surface area in the folded state. This mutant nonetheless folds very rapidly, and has the non-exponential folding kinetics of an incipient downhill folder even at the unfolding midpoint (k(m)(-1) approximately 2 micros, k(a)(-1)=15 micros at 56 degrees C). We also compare the thermodynamic melting transition of lambdaHG with the nominal two-state folding mutant lambdaQG, which has a similar melting temperature. Unlike lambdaQG, lambdaHG yields fluorescence wavelength-dependent cooperativities and probe-dependent melting temperatures. This result combined with previous work shows that the energy landscapes of lambda repressor mutants support all standard folding mechanisms.     

A comparative study of the unfolding thermodynamics of vertebrate metmyoglobins

Differential scanning microcalorimetry (DSC) of horse, rat, opossum, raccoon, carp, and armadillo metmyoglobins at alkaline pH gave data that fit the two-state unfolding model well. Monte Carlo studies were used to assess the impact of truncating DSC scans on the reliability of the calculated results when aggregation exotherms overlapped the unfolding endotherm at the high-temperature end of the scan. The DSC estimates for the conformational free energy at pH 8 and 298 K are compared to earlier results from isothermal acid and guanidinium chloride unfolding. Stability estimates at pH 8 for these six metmyoglobins obtained by DSC experiments do not agree with free energy estimates at pH 8 from guanidinium chloride unfolding. This is true for all three models used to extrapolate the free energy change to 0 M guanidinium chloride. Among these six myoglobins, significant variation appears in the temperature at which the myoglobin is half-unfolded, in the change in heat capacity upon unfolding, and in the change in enthalpy at 310 K. Calculations made with the hydrophobic model for protein folding [Baldwin, R.L. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8069] suggest that a sizable variation exists for that portion of the unfolding enthalpy change assigned to forces other than the hydrophobic effect.     

Folding of the multidomain ribosomal protein L9: the two domains fold independently with remarkably different rates

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (Tm = 67 degrees C for the C-terminal domain, 80 degrees C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72 degrees C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69 degrees C were used to determine the folding rate constant, 3.3 s-1, and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1, respectively, for the N-terminal domain at 25 degrees C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N- and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.     

Kinetic analysis of folding and unfolding the 56 amino acid IgG-binding domain of streptococcal protein G.

The 56 amino acid B domain of protein G (GB) is a stable globular folding unit with no disulfide cross-links. The physical properties of GB offer extraordinary flexibility for evaluating the energetics of the folding reaction. The protein is monomeric and very soluble in both folded and unfolded forms. The folding reaction has been previously examined by differential scanning calorimetry (Alexander et al., 1992) and found to exhibit two-state unfolding behavior over a wide pH range with an unfolding transition near 90 degrees C (GB1) at neutral pH. Here, the kinetics of folding and unfolding two naturally occurring versions of GB have been measured using stopped-flow mixing methods and analyzed according to transition-state theory. GB contains no prolines, and the kinetics of folding and unfolding can be fit to a single, first-order rate constant over the temperature range of 5-35 degrees C. The major thermodynamic changes going from the unfolded state to the transition state are (1) a large decrease in heat capacity (delta Cp), indicating that the transition state is compact and solvent inaccessible relative to the unfolded state; (2) a large loss of entropy; and (3) a small increase in enthalpy. The most surprising feature of the folding of GB compared to that of previously studied proteins is that its folding approximates a rapid diffusion controlled process with little increase in enthalpy going from the unfolded to the transition state.     

Refolding rate of stability-enhanced cytochrome c is independent of thermodynamic driving force.

N52I iso-2 cytochrome c is a variant of yeast iso-2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49-56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a "global suppressor" of destabilizing mutations. The equilibrium m-value for denaturant-induced unfolding of N52I iso-2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso-2 have been measured by stopped-flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild-type protein, iso-2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso-2 and N52I iso-2, despite the greater thermodynamic stability of N52I iso-2. Thus, there is no linear free-energy relationship between mutation-induced changes in stability and observable refolding rates. However, for N52I iso-2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso-2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two-state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two-state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes ("burst phase") are observed for both N52I iso-2 and iso-2 refolding. The "burst phase" amplitude is larger for N52I iso-2 than for iso-2, suggesting that the intermediates formed during the "burst phase" are stabilized by the N52I substitution.     

Influence of transition rates and scan rate on kinetic simulations of differential scanning calorimetry profiles of reversible and irreversible protein denaturation.

The thermodynamic parameters characterizing protein folding can be obtained directly using differential scanning calorimetry (DSC). They are meaningful only for reversible unfolding at equilibrium, which holds for small globular proteins; however, the unfolding or denaturation of most large, multidomain or multisubunit proteins is either partially or totally irreversible. The simplest kinetic model describing partially irreversible denaturation requires three states: Formula [see text] We obtain numerical solutions for N, U, and D as a function of temperature for this model and derive profiles of excess specific heat (Cp) in terms of the reduced variables v/ki and k1/k3, where v is the scan rate. The three-state model reduces to the two-state reversible or irreversible models for very large or very small values of k1/k3, respectively. The apparent transition temperature (Tapp) is always reduced by the irreversible step (U-->D). For all values of k3, Tapp is independent of v/k1 at sufficiently slow scan rates, even when denaturation is highly irreversible, but increases identically for all models at fast scan rates in which case the excess specific heat profile is determined by the rate of unfolding. Accurate values of delta H and delta S can be obtained for the reversible step only when k1 is more than 2000-50,000 times greater than k3. In principle, approximate values for the ratio k1/k3 can be obtained from plots of fraction unfolded vs fraction irreversibly denatured as a function of temperature; however, the fraction irreversibly denatured is difficult to measure accurately by DSC alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability and folding rates of domains spanning the large A-band super-repeat of titin

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.