Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes

Neuweiler, Hannes; Sharpe, Timothy D.; Johnson, Christopher M.; Teufel, Daniel P.; Ferguson, Neil; Fersht, Alan R.

doi:10.1016/j.jmb.2008.12.056

Cited by 30 publications

(77 citation statements)

References 44 publications

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“…Solvent viscosity and temperature-dependent measurements revealed that intrachain interactions are already present at the very early stage of folding. The results presented here are consistent with previous conclusions of barrier-limited, apparent 2-state folding of BBL as shown by a battery of ensemble-spectroscopic experiments (10)(11)(12)30) and very recent single-molecule FRET measurements that directly detected distinct native and denatured states (31).…”

supporting

confidence: 92%

“…The kinetics of BBL folding was further independent of site and nature of the probe and the spectroscopy applied, underscoring the cooperative nature of the folding process. Rate constants were also in good agreement with values extrapolated from denaturant-dependent folding kinetics (chevron analysis) measured by Trp fluorescence (10,11). We can assign the 10-s relaxation with confidence to the overall folding of the protein by comparison to the T-jump measurements because the ratio of rate constants from the ensemble kinetics give equilibrium constants that are in excellent agreement with those measured directly at equilibrium throughout the chemically induced denaturation transition (10).…”

Section: Observation Of Ultrafast Folding Kinetics In Single Bbl Molesupporting

confidence: 71%

“…We tested for the presence of possible perturbations introduced by protein modification, which could compromise conclusions deduced for wild-type protein. The introduction of individual Trp residues at positions 166 (H166W) and 142 (H142W) in the sequence does not induce any detectable energetic or structural perturbations (10)(11)(12). Chemical and thermal denaturation of AttoOxa11-modified BBL monitored using far-UV circular dichroism (CD) showed that AttoOxa11 does not induce significant perturbations either (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…All chemicals were purchased from Sigma-Aldrich and indicated if otherwise. Recombinant mutants of BBL were overexpressed in Escherichia coli and purified using affinity, ion exchange, and size-exclusion chromatography as described (10). Proteins were modified with the thiol-reactive (maleimide chemistry) oxazine fluorophore AttoOxa11 (Atto-Tec) at cysteine side chains introduced at positions 130 (A130C) and 167 (L167C).…”

Section: Methodsmentioning

confidence: 99%

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Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Neuweiler

Johnson

Fersht

2009

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

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116

133

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Single-molecule fluorescence resonance energy transfer (smFRET) experiments are extremely useful in studying protein folding but are generally limited to time scales of greater than Ϸ100 s and distances greater than Ϸ2 nm. We used single-molecule fluorescence quenching by photoinduced electron transfer, detecting short-range events, in combination with fluorescence correlation spectroscopy (PET-FCS) to investigate folding dynamics of the small binding domain BBL with nanosecond time resolution. The kinetics of folding appeared as a 10-s decay in the autocorrelation function, resulting from stochastic fluctuations between denatured and native conformations of individual molecules. The observed rate constants were probe independent and in excellent agreement with values derived from conventional temperaturejump (T-jump) measurements. A submicrosecond relaxation was detected in PET-FCS data that reported on the kinetics of intrachain contact formation within the thermally denatured state. We engineered a mutant of BBL that was denatured under the reaction conditions that favored folding of the parent wild type (''D phys''). D phys had the same kinetic signature as the thermally denatured state and revealed segmental diffusion with a time constant of intrachain contact formation of 500 ns. This time constant was more than 10 times faster than folding and in the range estimated to be the ''speed limit'' of folding. D phys exhibited significant deviations from a random coil. The solvent viscosity and temperature dependence of intrachain diffusion showed that chain motions were slaved by the presence of intramolecular interactions. PET-FCS in combination with protein engineering is a powerful approach to study the early events and mechanism of ultrafast protein folding.fluorescence correlation spectroscopy ͉ intrachain diffusion ͉ photoinduced electron transfer ͉ protein folding ͉ speed limit S ingle-molecule spectroscopy detects molecular heterogeneities that are hidden in the ensemble average of conventional spectroscopy and provides complementary insights into the conformationally heterogeneous process of protein folding (1, 2). Commonly applied single-molecule fluorescence resonance energy transfer (smFRET) methods have limitations in revealing conformational changes of less than Ϸ2 nm or measuring very fast processes occurring in under Ϸ1 ms because of inherently low fluorescence photon statistics and interference from photophysical effects. The investigation of small and ultrafast folding protein domains, which often represent the building blocks of larger assemblies, is of particular importance to our fundamental understanding of folding. Importantly, their time scales of conformational motions can overlap with those of atomistic computer simulations of protein folding pathways (3).Current limitations of smFRET methods can be overcome using new developments in fluorescence correlation spectroscopy (FCS). FCS measures the kinetics of stochastic fluorescence fluctuations arising from individual fluorescent molecul...

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supporting

confidence: 92%

Section: Observation Of Ultrafast Folding Kinetics In Single Bbl Molesupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Neuweiler

Johnson

Fersht

2009

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

Self Cite

116

133

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“…It is extremely difficult to falsify unimodal folding because most equilibrium and kinetic data can be interpreted by numerous alternative mechanisms, especially when the rate of interconversion of D and N states is on a faster time scale than that of observation (6). The dependence of rates of folding and unfolding on denaturant concentration is good evidence for the nature of the mechanism-classical chevron plots with a steep limb are compatible with only a folding scenario with a free energy barrier and a transition state, so there is a significant change of solvent-accessible surface between the ground and transition states (6)(7)(8). That evidence combined with a rationalization of the anomalous behavior of BBL has been used as evidence that the folding of BBL can be accommodated by a barrier limited folding model, with some heterogeneity of the native state (9)(10)(11)(12).…”

mentioning

confidence: 98%

Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer

Huang

Ying

Fersht

2009

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

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One controversial area in protein folding mechanisms is whether some small, ultra-fast-folding proteins exist in distinct native and denatured state ensembles, separated by an energy barrier, or if there is a continuum of states between native and denatured. In theory, the simplest way of distinguishing between single-state barrierless or ''downhill'' folding and conventional separate state folding is by single-molecule spectroscopy, which can detect either distinct populations of proteins or a continuum. But, the time resolution of approximately 1 ms of most confocal fluorescence microscopes for single-molecule fluorescence resonance energy transfer (SM-FRET) is longer than that for the structural relaxation of proteins such as BBL, whose mechanism of folding is controversial. We have constructed a highly sensitive confocal fluorescence microscope and measured the distribution of . The key protein in the unimodal hypothesis is BBL, a member of the peripheral subunit binding domain family, which is claimed to be a global downhill folder based especially on the dispersion of melting temperature for individual residues (2). It is extremely difficult to falsify unimodal folding because most equilibrium and kinetic data can be interpreted by numerous alternative mechanisms, especially when the rate of interconversion of D and N states is on a faster time scale than that of observation (6). The dependence of rates of folding and unfolding on denaturant concentration is good evidence for the nature of the mechanism-classical chevron plots with a steep limb are compatible with only a folding scenario with a free energy barrier and a transition state, so there is a significant change of solvent-accessible surface between the ground and transition states (6-8). That evidence combined with a rationalization of the anomalous behavior of BBL has been used as evidence that the folding of BBL can be accommodated by a barrier limited folding model, with some heterogeneity of the native state (9-12). Direct observation of distinct states of BBL in the transition region at equilibrium would provide compelling evidence for barrier limited folding (6, 13). The current method of choice is single-molecule fluorescence resonance energy transfer (SM-FRET) experiments, which has been widely applied in the studies of protein folding, structure, and function, and is especially useful in detection of heterogeneity of populations (14-21).We have applied SM-FRET to the B-domain of Protein A (BDPA) and directly observed the denatured and native states of this protein in its transition region (6), the signature of barrier-limited folding. We were not able to use SM-FRET to resolve unambiguously the folding mechanism of BBL because it folds in a fraction of a millisecond at room temperature, whereas the typical time resolution of SM-FRET was, until very recently, typically 1 ms (6,21,22), in which time BBL would rapidly equilibrate and appear as a time-averaged structure of the separate states if it folds by a barrier limited transition (23, 2...

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An in Vitro Cytomimetic of In‐Cell RNA Folding

Yoo

Davis

2022

ChemBioChem

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To discover the cytomimetic that accounts for cytoplasmic crowding and sticking on RNA stability, we conducted a twodimensional scan of mixtures of artificial crowding and sticking agents, PEG10k and M-PER TM . As our model RNA, we investigate the fourU RNA thermometer motif of Salmonella, a hairpinstructured RNA that regulates translation by unfolding and exposing its ribosome binding site (RBS) in response to temperature perturbations. We found that the addition of artificial crowding and sticking agents leads to a stabilization and destabilization of RNA folding, respectively, through the excluded volume effect and surface interactions. FRET-labels were added to the fourU RNA and Fast Relaxation Imaging (FReI), fluorescence microscopy coupled to temperature-jump spectroscopy, probed differences between folding stability of RNA inside single living cells and in vitro. Our results suggest that the cytoplasmic environment affecting RNA folding is comparable to a combination of 20 % v/v M-PER TM and 150 g/L PEG10k.

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Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes

Cited by 30 publications

References 44 publications

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Direct observation of ultrafast folding and denatured state dynamics in single protein molecules

Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer

An in Vitro Cytomimetic of In‐Cell RNA Folding

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