Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy

Kuwajima, Kunihiro; Garvey, Edward P.; Finn, Bryan E.; Matthews, C. Robert; Sugai, Shintaro

doi:10.1021/bi00245a005

Cited by 160 publications

(183 citation statements)

References 46 publications

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“…3A) and it probably originates from an altered environment of the tryptophan and/or of tyrosine residues at the stage of the folding intermediate (Kiefhaber et al, 1990~). Ordering of aromatic residues can contribute significantly to the CD of native proteins near 230 nm (Adler et al, 1973;Manning & Woody, 1989;Khan et al, 1989;Kuwajima et al, 1991). Spectra recorded after 20 s and after 10 min of refolding, when the additional formation of the intermediate via the slow reaction (7 = 170 s) is complete (cf.…”

Section: Time Course Of the Folding Intermediatementioning

confidence: 99%

Structure of a rapidly formed intermediate in ribonuclease T1 folding

et al. 1992

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Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single a-helix of ribonuclease TI. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the &sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (7 = 6,500 s at pH 5.0 and 10 "C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties. Keywords

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Section: Time Course Of the Folding Intermediatementioning

confidence: 99%

Structure of a rapidly formed intermediate in ribonuclease T1 folding

et al. 1992

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“…Stopped-flow circular dichroism studies showed that a large amount of secondary structure is formed in the dead time (milliseconds time range) of the observation. This has been observed for proteins such as ␣-lactalbumin and lysozyme (5), dihydrofolate reductase (6), and holocytochrome c (7). On the other hand, when using pulsed proton exchange followed by NMR identification of the protected protons, the formation of stable secondary structure elements could be detected over a slower observable time range (8,9).…”

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confidence: 92%

Kinetics of Secondary Structure Recovery during the Refolding of Reduced Hen Egg White Lysozyme

Roux¹,

Delepierre²,

Goldberg³

et al. 1997

Journal of Biological Chemistry

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We have shown previously that, in less than 4 ms, the unfolded/oxidized hen lysozyme recovered its native secondary structure, while the reduced protein remained fully unfolded. To investigate the role played by disulfide bridges in the acquisition of the secondary structure at later stages of the renaturation/oxidation, the complete refolding of reduced lysozyme was studied. This was done in a renaturation buffer containing 0.5 M guanidinium chloride, 60 M oxidized glutathione, and 20 M reduced dithiothreitol, in which the aggregation of lysozyme was minimized and where a renaturation yield of 80% was obtained. The refolded protein could not be distinguished from the native lysozyme by activity, compactness, stability, and several spectroscopic measurements. The kinetics of renaturation were then studied by following the reactivation and the changes in fluorescence and circular dichroism signals. When bi-or triphasic sequential models were fitted to the experimental data, the first two phases had the same calculated rate constants for all the signals showing that, within the time resolution of these experiments, the folding/oxidation of hen lysozyme is highly cooperative, with the secondary structure, the tertiary structure, and the integrity of the active site appearing simultaneously.Since Anfinsen's work on the in vitro renaturation of unfolded ribonuclease A (1), it is commonly accepted that all the information required for a protein to fold properly is contained in its amino acid sequence. However, the code that allows the formation of a fully folded protein from its amino acid sequence has not yet been deciphered. Three models are currently proposed to describe this process. The framework model is a sequential model in which secondary structure elements form first followed by a tighter packing of the molecule (2). Another model assumes that the polypeptide chain undergoes a rapid collapse driven by hydrophobic forces that would yield an intermediate close to the molten globule (3). In the puzzle model (4) structural elements form at different sites on the polypeptide chain, and their formation induces further folding of the whole protein.According to experimental data, it is not yet possible to determine which model most accurately describes the folding mechanism. However, some general features of protein folding have arisen. Stopped-flow circular dichroism studies showed that a large amount of secondary structure is formed in the dead time (milliseconds time range) of the observation. This has been observed for proteins such as ␣-lactalbumin and lysozyme (5), dihydrofolate reductase (6), and holocytochrome c (7). On the other hand, when using pulsed proton exchange followed by NMR identification of the protected protons, the formation of stable secondary structure elements could be detected over a slower observable time range (8, 9). Using both techniques to observe the folding of the same protein, as was the case for cytochrome c (7), lysozyme (10, 11), and interleukin-1␤ (12), secondary structures coul...

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“…1-5 Earlier studies have revealed that small globular proteins often populate compact intermediates with varying degrees of secondary structure within milliseconds of initiating the refolding reaction, long before they encounter the rate-limiting step of the folding reaction (e.g., refs. [6][7][8][9]. Direct observation of the formation of these early intermediates has, however, been difficult mainly due to the limited temporal resolution of conventional mixing devices.…”

Section: Introductionmentioning

confidence: 99%

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

Maki¹,

Cheng²,

Долгих³

et al. 2007

Journal of Molecular Biology

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To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophans in an apolar environment. The kinetics of folding was observed by continuous-and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophans in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (< 1 ms), indicating that this region of the protein undergoes a rapid specific collapse and remains uncoupled from the rest of the α-helical domain until the late stages of folding. For several variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophans reach their buried native environment only during the late stages of folding. Other variants show more complex behavior with a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophans into close contact with intramolecular quenchers.

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Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy

Cited by 160 publications

References 46 publications

Structure of a rapidly formed intermediate in ribonuclease T1 folding

Structure of a rapidly formed intermediate in ribonuclease T1 folding

Kinetics of Secondary Structure Recovery during the Refolding of Reduced Hen Egg White Lysozyme

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

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