Biomedical subjects

T Kiefhaber

Publications and source records attributed to T Kiefhaber.

At least 19 recordsLinked to original sources

PubMed2006

On the unusual fluorescence properties of xanthone in water.

Photo-excited xanthone is known to undergo ultrafast intersystem crossing (ISC) in the 1 ps time domain. Correspondingly, its fluorescence quantum yield in most solvents is very small ( approximately 10(-4)). Surprisingly, the quantum yield in water is 100 times larger, while ISC is still rapid ( approximately 1 ps), as seen by ultrafast pump probe absorption spectroscopy. Temperature dependent steady state and time resolved fluorescence experiments point to a delayed fluorescence mechanism, where the triplet (3)npi* state primarily accessed by ISC is nearly isoenergetic with the photo-excited (1)pipi* state. The delayed fluorescence of xanthone in water decays with a time constant of 700 ps, apparently by internal conversion between the (3)npi* state and the lowest lying triplet state (3)pipi*.

Luminescence↗

PubMed2001

Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments.

Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (<1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I(1)) with native-like secondary structure in the alpha-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I(1) and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I(2)). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I(1) have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.

Amides↗

PubMed2001

Apparent two-state tendamistat folding is a sequential process along a defined route.

The small all-beta-sheet protein tendamistat folds and unfolds rapidly in apparent two-state reactions. Kinetic measurements of two tendamistat variants under various solvent conditions reveal, however, that folding occurs in at least two sequential steps through a metastable obligatory intermediate. Depending on the solvent conditions either step can become rate limiting. The activation parameters indicate that the first step represents an enthalpic barrier whereas the second step is an entropic barrier at 25 degrees C. Our results suggest that initial non-specific collapse precedes formation of native secondary and tertiary structure in tendamistat folding. This points at a distinct route in tendamistat folding and indicates that partially folded metastable intermediates might play an important role in the mechanism of apparent two-state folding.

Circular Dichroism↗

PubMed2001

Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics.

Folding of tendamistat, an inhibitor of alpha-amylase, is a fast two-state process accompanied by two minor slow reactions, which were assigned to prolyl isomerization. In a proline-free variant, 5% of the molecules still fold slowly with a rate constant of 2.5 s(-1). This reaction is caused by a slow equilibrium between two populations of unfolded molecules. The time constant for this equilibration process, its sensitivity to LiCl and its temperature dependence identify it as a cis-trans isomerization of nonprolyl peptide bonds. Although nonprolyl peptide bonds have the cis conformation populating only approximately 0.15% in unfolded proteins, their large number generates a significant fraction of slow-folding molecules. This emphasizes that heterogeneous populations in an unfolded protein can induce complex folding kinetics on various time scales.

Amino Acid Substitution↗

PubMed2000

Denaturant-induced movement of the transition state of protein folding revealed by high-pressure stopped-flow measurements.

The small all-beta protein tendamistat folds and unfolds with two-state kinetics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure between 0.1 and 100 MPa (1 to 1, 000 bar). GdmCl-induced equilibrium unfolding transitions reveal that the volume of the native state is increased by 41.4 +/- 2.0 cm(3)/mol relative to the unfolded state. This value is virtually independent of denaturant concentration. The use of a high-pressure stopped-flow instrument enabled us to measure the activation volumes for the refolding (DeltaVo/f) and unfolding reaction (DeltaVo/u) over a broad range of GdmCl concentrations. The volume of the transition state is 60% native-like (DeltaVo/f) = 25.0 +/- 1.2 cm(3)/mol) in the absence of denaturant, indicating partial solvent accessibility of the core residues. The volume of the transition state increases linearly with denaturant concentration and exceeds the volume of the native state above 6 M GdmCl. This result argues for a largely desolvated transition state with packing deficiencies at high denaturant concentrations and shows that the structure of the transition state depends strongly on the experimental conditions.

Guanidine↗

PubMed1999

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.

Animals↗

PubMed1999

The speed limit for protein folding measured by triplet-triplet energy transfer.

A direct measure of intramolecular chain diffusion is obtained by the determination of triplet-triplet energy-transfer rates between a donor and an acceptor chromophore attached at defined points on a polypeptide chain. Single exponential kinetics of contact formation are observed on the nanosecond time scale for polypeptides in which donor and acceptor are linked by repeating units of glycine and serine residues. The rates depend on the number of peptide bonds (N) separating donor and acceptor and show a maximum for the shortest peptides (N = 3) with a time constant (tau = 1/k) of 20 ns. This sets an upper limit for the speed of formation of the first side-chain contacts during protein folding.

Energy Transfer↗

PubMed1999

Intermediates can accelerate protein folding.

The effect of intermediates on the rate of protein folding is explored by applying Kramers' theory of diffusive barrier crossing in the high friction limit. Intermediates are represented as local minima in the transition barrier. We observe that very large or very small additional barriers created by the intermediates slow down the folding process. The rate of folding markedly increases, however, when the additional barriers become >1 kBT but leave the overall barrier height unchanged. This rate-enhancing effect is caused by a favorable entropic contribution to the free energy of activation, and it increases with the number of intermediates up to a limiting value. From these calculations, we conclude that optimized transition barriers should contain partially folded high energy intermediates.

Animals↗

PubMed1999

Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering.

We have used synchrotron radiation, together with stopped-flow and continuous-flow mixing techniques to monitor refolding of lysozyme at pH 5.2. From data measured at times which range from 14 ms to two seconds, we can monitor changes in the size, the shape and the pair distribution function of the polypeptide chain during the folding process. Comparison of the results with the properties of native and GdmCl-unfolded lysozyme shows that a major chain collapse occurs in the dead-time of mixing. During this process about 50 % of the change in radius of gyration between the unfolded protein and the native state occurs and the polypeptide chain adopts a globular shape. Time-resolved fluorescence spectra of this collapsed state suggest that the hydrophobic side-chains are still highly solvent accessible. A subsequently formed intermediate with helical structure in the alpha-domain is nearly identical in size and shape with native lysozyme and has a solvent-inaccessible hydrophobic core. Despite its native-like properties, this intermediate is only slightly more stable (DeltaG0=-4 kJ/mol) than the collapsed state and still much less stable than native lysozyme (DeltaDeltaG0=36 kJ/mol) at 20 degrees C.

Animals↗

PubMed1998

Stability of trimeric OmpF porin: the contributions of the latching loop L2.

The channel-forming protein OmpF porin from Escherichia coli spans the bacterial outer membrane. Each of the three monomers comprises a hollow, 16-stranded beta-barrel. These are associated to homotrimers which are unusually stable, due mostly to hydrophobic interactions between the beta-barrels. In addition, a loop, L2 connects one subunit to its neighbor by latching into its channel. Residue E71 on loop 2 is integrated into an ionic network and forms salt bridges and hydrogen bonds with R100 and R132 on the channel wall in the adjacent subunit. To examine these contributions quantitatively, six single-site, two double, and one deletion mutant were constructed on the basis of the atomic coordinates of the protein. Differential scanning calorimetric analysis showed that the salt-bridge, E71-R100, contributes significantly to trimer stability: the substitution E71Q causes a decrease of the transition temperature from 72 to 48 degreesC, with DeltaHcal diminishing from 430 to 201 kcal mol-1. A nearby substitution in the loop, D74N, has lesser effects on thermal stability, while the deletion in L2 (Delta69-77) has an effect comparable to that of E71Q. X-ray structure analysis to 3.0 A resolution revealed only local structural differences in the mutants except for the substitution R100A, where another residue, R132, is found to fill the gap left by the truncated side chain of A100. Functional assays in planar lipid bilayers show significantly increased cation selectivities if the charge distribution was affected.

Alanine↗

PubMed1998

Kinetics of lysozyme refolding: structural characterization of a non-specifically collapsed state using time-resolved X-ray scattering.

We report time-resolved small angle X-ray scattering (SAXS) studies of the structural characteristics of the collapsed state of lysozyme from henegg white (HEL) obtained on initiating refolding by rapidly changing solvent conditions from 8 M to 1.1 M urea at pH 2.9. At this reduced pH the lifetime, of about one second, of the non-specifically collapsed ensemble is considerably prolonged relative to its value at pH 5.2. The SAXS studies are combined with time resolved measurements of tryptophan fluorescence and of the rate of formation of native molecules using interrupted refolding experiments. We observe large burst phase changes in intrinsic tryptophan fluorescence and in the radius of gyration (Rg) which is reduced from 22 A in the fully unfolded state to approximately 19 to 20 A. Subsequent decrease of the Rg to the value for native lysozyme (15 A) follows the time course of formation of native molecules. Single exponential fits to the singular value decomposition (SVD) components of the SAXS data allow reconstruction of the SAXS profile at early time points of refolding. The results of this analysis suggest a globular shape of the collapsed state. A similar fit to the forward scattering amplitude, I(0), suggests that the collapsed state has a solvent accessible surface area which is considerably increased relative to that of the native protein. These results show directly that the non-specifically collapsed state formed during the burst phase in lysozyme refolding indeed represents a molecular compaction and a change in shape from a fully denatured random coil state (albeit restricted by disulfide bonds) to an ensemble of globular conformations which, however, have not yet formed a solvent-protected hydrophobic core.

Animals↗

PubMed1997

Effect of preformed correct tertiary interactions on rapid two-state tendamistat folding: evidence for hairpins as initiation sites for beta-sheet formation.

The role of preformed correct side chain interactions, such as disulfide bonds, on protein folding kinetics is still not well understood. We investigated the effect of disulfide bond replacements on folding and stability of the small beta-sheet protein tendamistat. Tendamistat folds very fast (tau = 10 ms at pH 7 in water) and without detectable intermediates, which facilitates molecular interpretation of the kinetic data. Tendamistat contains two disulfide bonds, one between cysteines 11 and 27, which connects the ends of a beta-hairpin, and a second one between cysteines 45 and 73, which brings together the two outer strands of a three-stranded beta-sheet. Two single-disulfide variants of the protein were prepared by site-directed mutagenesis (tendamistat C11A/C27S and tendamistat C45A/C73A), and the effects on stability and on folding were monitored. Replacement of either disulfide bond leads to a large decrease in protein stability (DeltaDeltaG0 = 6.0 kcal/mol for the C11A/C27S variant and 5.1 kcal/mol for the C45A/C73A variant). This effect is caused both by entropic stabilization of the unfolded state and by enthalpic destabilization of the native structure. Kinetic experiments show that the main effect of fixed side chain contacts is on the unfolding rate. For both single-disulfide variants, unfolding is strongly accelerated (4250 times in the C11A/C27S variant and 250 times in the C45A/C73A variant) whereas the refolding rate constants are only slightly decreased. The activation parameters show that the observed small effect on the refolding reaction in the C11A/C27S variant is a consequence of large and compensating changes in the entropy and enthalpy of activation. Structural interpretation of the kinetic data suggests that formation of the beta-hairpin stabilized by the C11-C27 disulfide bond forms in the rate-limiting step of the refolding process. The interactions between the outer strands of the beta-sheet connected by the C45-C73 disulfide bond, in contrast, are made late in refolding. These results support the idea that beta-hairpins are initiation sites for beta-sheet formation and that additional strands are added late in the folding process.

Disulfides↗

PubMed1997

Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate.

We investigated the role of a partially folded intermediate that transiently accumulates during lysozyme folding. Previous studies had shown that the partially folded intermediate is located on a slow-folding pathway and that an additional fast direct pathway from the unfolded state to the native state exists. Kinetic double-jump experiments showed that the two folding pathways are not caused by slow equilibration reactions in the unfolded state. Rather, kinetic partitioning occurs very early in lysozyme refolding, giving the molecules the chance to enter the direct pathway or a slow-folding channel. Fitting the guanidinium chloride dependencies of the refolding and unfolding reactions to analytical solutions for different folding scenarios enables us to propose a triangular mechanism as the minimal model for lysozyme folding explaining all observed kinetic reactions: [diagram in text]. All microscopic rate constants and their guanidinium chloride dependencies could be obtained from the experimental data. The results suggest that population of the intermediate during refolding increases the free energy of activation of the folding process. This effect is due to the increased stability of the intermediate state compared to the unfolded state leading to an increase in the free energy of activation (deltaG0) compared to folding in the absence of populated intermediate states. The absolute energy of the transition state is identical on both pathways. The results imply that pre-formed secondary structure in the folding intermediate obstructs formation of the transition state of folding but does not change the nature of the rate-limiting step in the folding process.

Animals↗

PubMed1997

Folding of the disulfide-bonded beta-sheet protein tendamistat: rapid two-state folding without hydrophobic collapse.

We investigated the reversible folding and unfolding reactions of the small 74 amino acid residue protein tendamistat. The secondary structure of tendamistat contains only beta-sheets and loop regions and the protein contains two disulfide bonds. Fluorescence-detected refolding kinetics of tendamistat (disulfide bonds intact) comprise of a major rapid fast reaction (tau = 10 ms in water) and two minor slow reactions. In the fast reaction 80% of the unfolded molecules are converted to native protein. The two slow reactions are part of a parallel slow folding pathway. On this pathway the rate-limiting step in the formation of native molecules is cis to trans isomerization of at least one of the three trans Xaa-Pro peptide bonds. This reaction is catalyzed efficiently by the enzyme peptidyl-prolyl cis-trans isomerase. Comparison of kinetic data with equilibrium unfolding transitions shows that the fast folding pathway follows a two-state process without populated intermediate states. Additionally, various sensitive tests did not detect any rapid chain collapse during tendamistat folding prior to the acquisition of the native three-dimensional structure. These results show that pre-formed disulfide bonds do not prevent efficient and rapid protein folding.

Amino Acid Isomerases↗

PubMed1997

Direct measurement of nucleation and growth rates in lysozyme folding.

A kinetic folding intermediate of hen lysozyme is shown to form in a nucleation/growth type of mechanism. Under native solvent conditions, a nucleated state is formed slowly during refolding (tau = 14 +/- 1 ms at 0 M GdmCl) and is rapidly converted to the folding intermediate (tau = 300 +/- 150 micros at 0 M GdmCl). Under these conditions the nucleated state represents a high-energy state compared to the folding intermediate (delta deltaG0 = 13.7 +/- 3 kJ/mol). At elevated concentrations of GdmCl, the nucleated state becomes more stable than the intermediate and it consequently becomes transiently populated during unfolding of the intermediate state. This allowed us to measure the rate constant of the growth step using stopped-flow double-jump experiments. At high concentrations of GdmCl (>5 M), the growth step becomes rate-limiting in unfolding, leading to the frequently observed rollover in the GdmCl dependence of the logarithm of the apparent rate constant of the unfolding reaction.

Animals↗

PubMed1996

Native-like beta-structure in a trifluoroethanol-induced partially folded state of the all-beta-sheet protein tendamistat.

The effect of trifluoroethanol (TFE) on the structure of the all-beta-sheet protein tendamistat was investigated. At low concentrations TFE induces cooperative loss of the native tertiary structure leading to a partially folded state. The loss of specific side-chain interactions in the transition from the native state of the TFE-induced state is demonstrated by the disappearance of the CD bands in the aromatic region, a reduced chemical shift dispersion of the one-dimensional 1H NMR spectrum and a broad, uncooperative thermal unfolding transition of the partially folded state. An increased line-width of the NMR bands in the TFE state compared with the unfolded state suggests the presence of multiple, rapidly interconverting conformations. Hydrogen-exchange studies of amide proteins in the TFE state reveal the existence of defined hydrogen bonds at the same locations as in the native state, but with largely reduced stability. This suggests the presence of most of the native beta-sheet structure. These results are supported by Fourier transformed IR measurements, which show nearly the same amount of beta-structure in the TFE state and in the native state. Far UV CD spectroscopy suggests the induction of some alpha-helical structure upon addition of TFE, which appears to be located mainly in regions corresponding to loops or random structure in the native state and which seems to represent fluctuating conformations with preferred backbone angles rather than stable, hydrogen-bonded alpha-helices. These results show that stable non-local interactions, as they occur in beta-sheets, can form in the absence of specific side-chain interactions. The presence of a subset of the native long-range interactions and the absence of stable non-native interactions suggests that the observed partially folded state might represent an early intermediate on a hierarchical folding pathway of tendamistat.

Hydrogen Bonding↗

PubMed1996

Hydrogen exchange and the unfolding pathway of ribonuclease A.

Recently, when the kinetic unfolding process of ribonuclease A was monitored by hydrogen exchange (T. Kiefhaber and R.L. Baldwin, Proc. Natl. Acad. Sci. USA, 92 (1995) 2657-2661), all peptide hydrogen bonds were found to undergo rapid exchange in a single kinetic step under conditions where unfolding is slow and the intrinsic rate of hydrogen exchange is fast (pH 8.0, 10 degrees C, 4.5 M guanidinium chloride). Comparison with the unfolding rate measured by circular dichroism indicates that hydrogen exchange is caused by the rate-limiting step of unfolding. No evidence was found for partly unfolded intermediates that are formed slowly enough to be observed by EX1 (unfolding-limited) hydrogen exchange. Some peptide NH protons were found to show, in addition to EX1 exchange, faster EX2 exchange that is base-catalyzed. The EX2 exchange is caused by species that equilibrate rapidly with the native protein at the start of the unfolding process. These species might include rapidly formed unfolding intermediates. We show here that any such unfolding intermediates must have large protection factors because the EX2 reactions of ribonuclease A under these unfolding conditions have protection factors > or = 2500.

Hydrogen↗

PubMed1996

A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions.

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.

Amino Acid Sequence↗