Dynamics of Unfolded Polypeptide Chains as Model for the Earliest Steps in Protein Folding

A light-switchable peptide is transformed with ultrashort pulses from a ␤-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular mechanics is used to interpret the absorption transients. Illumination of the ␤-hairpin state triggers an unfolding reaction that visits several intermediates and reaches the unfolded state within a few nanoseconds. In this unfolding reaction to the equilibrium hydrophobic cluster conformation, the system does not meet significant barriers on the free-energy surface. The reverse folding process takes much longer because it occurs on the time scale of 30 s. The folded state has a defined structure, and its formation requires an extended search for the correct hydrogen-bond pattern of the ␤-strand.density functional theory calculation ͉ peptide folding ͉ TrpZip2 ͉ ultrafast infrared spectroscopy F olding is a key process during the formation of a functional protein after the synthesis of its amino acid chain (1). During folding, the amino acid chains are rearranged in highly complex processes, for which a detailed understanding is still missing. Straightforward solutions of the folding problem are prevented by the high dimensionality of the protein conformational spaces and by the wide range of relevant time scales. Thus, for complexity reduction, one has to focus on typical protein substructures, which are small enough to be analyzed by present-day technology. With corresponding peptide model systems there is a chance to monitor the formation of secondary structures, to identify characteristic intermediate states of these folding dynamics, and to carry out realistic simulations on a molecular level. An interesting class of model systems are light-triggered peptides, within which the incorporation of a photoresponsive element can initiate structural changes.Among corresponding photoresponsive chromophores, azobenzene derivatives have become a popular choice when it comes to selecting a fast conformational trigger for peptide refolding, because their structure significantly changes on time scales of a few hundred femtoseconds after photoexcitation. Indeed, an azobenzene chromophore, used as a backbone element within cyclic peptides, was shown to induce large-scale conformational changes, whose dynamics could be monitored by visible (2) and IR (3) spectroscopy. Moreover, the experimental results clearly showed that the initial, strongly driven conformational changes of the peptide proceeded within a few picoseconds after the trigger event. Externally linked to an ␣-helix, azobenzene also was used for unfolding (4) and refolding (5) of an ␣-helical model peptide in the nanosecond to microsecond time range.A prominent secondary structure motif is the ␤-sheet, for which a ␤-hairpin represents a minimal model (6, 7). We and others (8-10) have recently focused on the design of photocontrolled ␤-hairpin peptides. To enable an ultrafa...

supporting

confidence: 54%

Light-triggered β-hairpin folding and unfolding

Schrader

Schreier

Cordes

et al. 2007

“…To gain insight into the structure and dynamics of unfolded proteins, we have recently studied the kinetics of intramolecular loop formation in model polypeptide chains with repetitive amino acid sequences and in sequences derived from natural proteins (18)(19)(20)(21). Using intramolecular triplet-triplet energy transfer (TTET) between a donor and an acceptor group, we observed a decreasing rate constant for loop formation, k c , with increasing denaturant concentration (22).…”

mentioning

confidence: 99%

End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation

Möglich

Joder

Kiefhaber

2006

Self Cite

234

335

Characterization of the unfolded state is essential for the understanding of the protein folding reaction. We performed time-resolved FRET measurements to gain information on the dimensions and the internal dynamics of unfolded polypeptide chains. Using an approach based on global analysis of data obtained from two different donor–acceptor pairs allowed for the determination of distance distribution functions and diffusion constants between the chromophores. Results on a polypeptide chain consisting of 16 Gly-Ser repeats between the FRET chromophores reveal an increase in the average end-to-end distance from 18.9 to 39.2 Å between 0 and 8 M GdmCl. The increase in chain dimensions is accompanied by an increase in the end-to-end diffusion constant from (3.6 ± 1.0) × 10 −7 cm 2 s −1 in water to (14.8 ± 2.5) × 10 −7 cm 2 s −1 in 8 M GdmCl. This finding suggests that intrachain interactions in water exist even in very flexible chains lacking hydrophobic groups, which indicates intramolecular hydrogen bond formation. The interactions are broken upon denaturant binding, which leads to increased chain flexibility and longer average end-to-end distances. This finding implies that rapid collapse of polypeptide chains during refolding of denaturant-unfolded proteins is an intrinsic property of polypeptide chains and can, at least in part, be ascribed to nonspecific intramolecular hydrogen bonding. Despite decreased intrachain diffusion constants, the conformational search is accelerated in the collapsed state because of shorter diffusion distances. The measured distance distribution functions and diffusion constants in combination with Szabo–Schulten–Schulten theory were able to reproduce experimentally determined rate constants for end-to-end loop formation.

“…In the case of type 2 residues, k op equals the rate constant associated with unfolding of native apoflavodoxin to a specific PUF, k N-PUF , and k cl equals the rate constant associated with refolding of the PUF in question (26). Comparison of the latter rate constants from the opening (i.e., unfolding) rate constants k op according to ⌬G op ‡ ϭ ϪRT ln(k op͞k0) using a value of 10 8 for k0 (28). The depth of the minimum in the free energy landscape in which I 2 resides is unknown and therefore is represented by a dashed line.…”

Section: Resultsmentioning

confidence: 99%

The folding energy landscape of apoflavodoxin is rugged: Hydrogen exchange reveals nonproductive misfolded intermediates

Bollen

Kamphuis

Mierlo

2006

Many native proteins occasionally form partially unfolded forms (PUFs), which can be detected by hydrogen͞deuterium exchange and NMR spectroscopy. Knowledge about these metastable states is required to better understand the onset of folding-related diseases. So far, not much is known about where PUFs reside within the energy landscape for protein folding. Here, four PUFs of the relatively large apoflavodoxin (179 aa) are identified. Remarkably, at least three of them are partially misfolded conformations. The misfolding involves side-chain contacts as well as the protein backbone. The rates at which the PUFs interconvert with native protein have been determined. Comparison of these rates with stopped-flow data positions the PUFs in apoflavodoxin's complex folding energy landscape. PUF1 and PUF2 are unfolding excursions that start from native apoflavodoxin but do not continue to the unfolded state. PUF3 and PUF4 could be similar excursions, but their rates of formation suggest that they are on a dead-end folding route that starts from unfolded apoflavodoxin and does not continue all of the way to native protein. All PUFs detected thus are off the protein's productive folding route.hydrogen͞deuterium exchange ͉ partially unfolded forms ͉ protein folding P rotein folding can be described by using a free energy landscape model (1, 2). In this model an unfolded protein molecule descends along a funnel describing its free energy until it reaches the state with the lowest free energy, which is the native state. The landscape often contains local minima, which host folding intermediates. Whereas the native state of many proteins is well characterized, little is known about the metastable intermediate states and their positions along the folding funnel. Knowledge about these metastable states is necessary to improve the understanding of protein folding and foldingrelated diseases, because rarely populated partially folded forms of proteins are known to initiate the formation of amyloids (3).Detection by nuclear magnetic resonance (NMR) spectroscopy of native state hydrogen͞deuterium exchange (H͞D exchange), i.e., in the presence of small amounts of a denaturant, allows the characterization of partially unfolded forms (PUFs) of a protein (4). These PUFs are interpreted to reside in highenergy minima in the folding energy landscape (5, 6). PUFs are often undetectable by other techniques, because, for example, they reside behind the highest-energy transition state for folding and because they do not populate significantly at equilibrium. Analysis of H͞D exchange data with models that statistically sample the conformational space accessible to a protein can give insight into the high-energy conformations that a protein can adopt (7-9). However, little evidence exists that links PUFs to the folding intermediates that populate during equilibrium unfolding, kinetic unfolding, or kinetic refolding of proteins. This missing link makes it difficult to position PUFs within the energy landscape for protein folding (10).Recently, bot...