The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

Teilum, Kaare; Poulsen, Flemming M.; Akke, Mikael

doi:10.1073/pnas.0509100103

Cited by 22 publications

(20 citation statements)

References 44 publications

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“…The highest-flux pathways connecting a fully extended state to the native state show contact formation between helices 1 and 4 that are coupled to the folding transition, consistent with phi-value analysis by Kragelund et al 1 (Figure 5b). Furthermore, our model predicts a near-native state with a displaced helix 3, corresponding well to a near-native intermediate identified by Teilum et al 2 .…”

Section: Resultssupporting

confidence: 86%

Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

et al. 2012

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Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A-binding protein (ACBP), a two-state folder (folding time ~10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenises, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond timescale. These studies, along with with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (~100 µs) timescale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov State Model (MSM) of the ACBP folding reaction, constructed from over 30 milliseconds of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure and kinetics consistent with experiment, but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate, but rather a more heterogeneous and slow acquisition of unfolded-state structure.

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Section: Resultssupporting

confidence: 86%

Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

et al. 2012

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“…Similarly, there is no obvious relation to the transition state structure (54). Our observation is in contrast to recent measurements on chymotrypsin inhibitor 2 and acyl-CoA-binding protein, where indications were found for a substantial deviation of the collapsed denatured state from Gaussian chain behavior (17) possibly involving folding intermediates (55). In summary, we conclude from the single-molecule fluorescence data that unfolded CspTm is close to a random coil in terms of polymer physics, even under near-physiological conditions.…”

Section: Discussioncontrasting

confidence: 56%

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Hoffmann

Kane

Nettels

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

215

338

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We have used the combination of single-molecule Fö rster resonance energy transfer and kinetic synchrotron radiation circular dichroism experiments to probe the conformational ensemble of the collapsed unfolded state of the small cold shock protein CspTm under near-native conditions. This regime is physiologically most relevant but difficult to access experimentally, because the equilibrium signal in ensemble experiments is dominated by folded molecules. Here, we avoid this problem in two ways. One is the use of single-molecule Fö rster resonance energy transfer, which allows the separation of folded and unfolded subpopulations at equilibrium and provides information on long-range intramolecular distance distributions. From experiments with donor and acceptor chromophores placed at different positions within the chain, we find that the distance distributions in unfolded CspTm agree surprisingly well with a Gaussian chain not only at high concentrations of denaturant, where the polypeptide chain is expanded, but also at low denaturant concentrations, where the chain is collapsed. The second, complementary approach is synchrotron radiation circular dichroism spectroscopy of collapsed unfolded molecules transiently populated with a microfluidic device that enables rapid mixing. The results indicate a ␤-structure content of the collapsed unfolded state of Ϸ20% compared with the folded protein. This suggests that collapse can induce secondary structure in an unfolded state without interfering with long-range distance distributions characteristic of a random coil, which were previously found only for highly expanded unfolded proteins.Gaussian chain ͉ microfluidic mixing ͉ protein folding ͉ random coil ͉ secondary structure W ith the discovery of small proteins that fold in the absence of populated intermediates (1), our quantitative understanding of the elementary properties of protein folding reactions has made significant advances, including the structural characterization of transition states for folding (2) and the prediction of folding rates from native structure (3-5). One of the most severe limitations for the further development of these approaches is our ignorance about the energetic or structural properties of unfolded † † states of proteins. Because of the structural heterogeneity and complexity of the ensembles of conformations populated by unfolded proteins, their experimental characterization has proven extremely difficult. Traditional methods, such as small-angle scattering techniques (6), provide only global physical properties, e.g., the radius of gyration. In some cases, more detailed structural information can be obtained from NMR (7-10), but these studies usually provide information about the denatured state only under nonnative conditions, typically in the presence of large concentrations of denaturant, or through severe destabilization of the native state induced by covalent modification or mutations. The most interesting and physiologically relevant situation, however, is that of an unfolded sta...

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“…Additional experimental and computational studies support these findings (5,(19)(20)(21)(22)(23)(24)(25)(26). In the present study, we show that a combination of site-directed mutagenesis and chemical shift analysis can be used to identify specific tertiary structure formation in the unfolded state of the four-helix protein, ACBP.ACBP is an intensively studied model system for the folding of four-helix bundle proteins (9,(15)(16)(17)(27)(28)(29)(30)(31)(32)(33)(34). Key residues for folding of ACBP have been described in a recent study (9), and residues that are rate determining for protein folding have been determined using Φ-value analysis (27, 31).…”

supporting

confidence: 53%

“…ACBP is an intensively studied model system for the folding of four-helix bundle proteins (9,(15)(16)(17)(27)(28)(29)(30)(31)(32)(33)(34). Key residues for folding of ACBP have been described in a recent study (9), and residues that are rate determining for protein folding have been determined using Φ-value analysis (27, 31).…”

mentioning

confidence: 99%

Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein

Bruun

Iešmantavičius

Danielsson

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. Some mutations change the chemical shifts of residues remote from the site of mutation indicating the presence of an interaction between the mutated and the remote residues, suggesting that the formation of helix segments and helix-helix interactions is cooperative. We can begin to track down the folding mechanism of this protein using only experimental data by combining the information available for the rate limiting structure formation during the folding process with measurements of the site specific hydrogen bond formation in the burst phase, and with the existence prior to the folding reaction of tertiary structures in the ensemble of otherwise unfolded structures observed in the present study.protein folding | transient structure formation P rotein folding is one of the fundamental processes in living organisms. Genes have been selected in evolution to code for peptide chains with amino acid sequences that fold into well-defined functional structures. Although selection and evolution have ensured that amino acid sequences and the protein folding processes are optimized in any organism, the nature of these mechanisms remains to be fully understood (1).Protein folding has been intensively studied for several decades (2), but so far the atomic details of the folding processes have not been determined experimentally for any protein. The accumulation of detailed knowledge about the mechanisms of protein folding pathways is the tedious and perhaps only way toward understanding the origin of the wealth of protein architectures, which are being formed in the courses of protein folding events. It is relevant, therefore, to develop methods that may describe both the kinetics and the equilibrium thermodynamics of the processes of protein folding. The kinetics of the global protein folding processes are known for many proteins to be very fast, and it has been studied primarily using rapid mixing techniques (3). However, studies of global protein folding processes do not provide any information about those underlying processes, which hold the key to understanding why and how a unique amino acid sequence can form regular protein architecture. Because many proteins do fold spontaneously, there must be an imbedded autonomy in the amino acid sequence directing the course and the result of folding.Here we are concerned with the detection of structure formation in the initial stage of a protein folding event. For this purpose we are using NMR spectroscopy to study the formation of interactions in the unfolded state of acyl coenzyme A binding protein (ACBP) between residues that are separated by more than 10 residues in the amino acid sequence. A number of studies suggest that the transiently populated structures in the unfolded state ...

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The inverted chevron plot measured by NMR relaxation reveals a native-like unfolding intermediate in acyl-CoA binding protein

Cited by 22 publications

References 44 publications

Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

Slow Unfolded-State Structuring in Acyl-CoA Binding Protein Folding Revealed by Simulation and Experiment

Mapping protein collapse with single-molecule fluorescence and kinetic synchrotron radiation circular dichroism spectroscopy

Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein

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