In contrast with intermediates that accumulate before the rate-limiting step, the barrier top species determine the overall folding rate and hold the keys to the mechanism. The major challenge is the timescale of the process, which needs to be longer than 0. Under these native conditions, the most populated non-native conformations are expected to be those that sit at the top of the shallow barrier that is found at the denaturation midpoint [ 55 ].
These RD-NMR experiments are arguably the first example of high-resolution structural analysis of the conformations that determine the folding rate and mechanism of a protein. Interestingly, the structural properties of the barrier top derived by RD-NMR were in very close agreement with the experimental analysis of the folding interaction network and the long-timescale MD simulations obtained independently on the same protein [ ] see the previous section. In addition to obtaining structural information, it is also important to measure dynamic events such as folding transitions and microscopic pathways because they reveal the heterogeneity of mechanisms.
For a barrier-crossing process, folding transition paths are the conformational excursions that take the protein over the barrier Figure 5. Transitions occur very rarely, but are extremely fast. In fact, the typical time the molecule spends crossing the barrier is related to the pre-exponential factor of eqn 1.
Therefore one would expect folding transition paths to take a few microseconds and be broadly distributed. Resolving individual transition paths requires methods that simultaneously reach single-molecule, sub-microsecond and atomic resolutions. Not only that, but also the observation times need to be sufficiently long to catch these rare events Figure 5. The folding free energy landscape of a single-domain protein is often represented with a simple 1D surface with two minima for the native N and unfolded states U , and a more or less pronounced barrier separating them left.
On this surface, individual molecules dwell on either minimum for most of the time since climbing the barrier is a probabilistically rare event. However, when it happens, the transitions across the barrier are fast because they are only limited by the conformational motions at the barrier top. This results in single-molecule trajectories that slowly alternate between U and N with very sharp transitions right. The average dwell times on U and N are equivalent to the inverse of the folding and unfolding rate constants measured in bulk kinetic experiments respectively, whereas the sharp transitions correspond to the barrier-crossing paths.
The first pass at estimating folding transition path times experimentally came once again from fast-folding proteins [ ]. Such a process, termed the molecular phase, should correspond to the depopulation of the barrier top in response to the perturbation. Eaton and co-workers have attempted to measure transition paths directly with single-molecule fluorescence methods to obtain estimates of the average folding transition path time for several proteins [ , ].
The time-resolution limitation was overcome by slowing down folding dynamics by addition of viscogens and analysing the single-molecule trajectories photon by photon [ 47 ]. Further analysis and comparison with MD simulations has revealed that the barrier crossing for this protein involves formation of off-register hydrogen bonds between the helices that need to break to proceed towards the native state, which increases internal friction and thus slows down the pre-exponential term [ ].
Despite the impressive advances in experimental methods described above, atomistic MD simulations are possibly the only practical approach to resolve folding transitions of individual molecules with the time and structural resolution required to derive mechanistic information. Fast folding has stimulated the development and benchmarking of various approaches based on MD simulations [ 59 , 63 , , — ]. Recently, Shaw et al. The simulations folded most of these proteins into their native structure multiple times and with rates similar to those determined experimentally [ ].
In the simulations, collapse and secondary structure occurred together to form a compact form in which a native-like topology was stabilized by a small subset of key long-range native contacts. The upper panels show two individual MD trajectories for each protein revealing multiple folding and unfolding events.
The lower panels show the 1D free energy surfaces derived from the MD simulations together with the superposition of the experimentally determined native structure red and the native structure identified by the simulations blue. Figure derived with permission from Lindorff-Larsen et al. The possibility of simulating multiple folding—unfolding transitions in single trajectories offers very exciting possibilities to investigate folding mechanisms in detail.
The case for defined protein folding pathways
The simulations provide the extreme resolution that experiments could never achieve, but still use approximate force fields to describe protein energetics and dynamics. Increasingly sophisticated experiments, such as those described in the present review, provide the critical benchmarks for further refinement of simulations in a perfect symbiosis.
A related area of interest focuses on the structural and functional analysis of IDPs [ ], especially after the realization that IDPs amount to a very large fraction of the proteome [ ].
These proteins exhibit structural disorder in native conditions and folding coupled to binding via complex mechanisms [ ]. Experimental studies have reported IDPs that bind to their partners through either induced fit or conformational selection mechanisms [ ]. Some IDPs bind to multiple partners that are structurally diverse [ ], a feature that allows them to moonlight [ ] or produce sophisticated allosteric effects [ ].
It turns out that IDPs and fast-folding proteins, especially one-state downhill folders, are closely interconnected. It has been noticed recently that folding rate, stability and co-operativity are intimately coupled so proteins that fold fast also unfold fast, are marginally stable and are minimally co-operative [ ]. In fact, the stability of domains identified as downhill folders seems to be poised towards exhibiting partial disorder under physiological conditions. This trend has been observed by investigating homologous fast-folding domains from meso-, thermo- and hyper-thermophilic organisms in which the denaturation temperature of the domain tracks the living temperature of the organism [ ].
So-called IDPs, on the other hand, have significant residual structure, as shown by NMR [ ] and single-molecule fluorescence [ ] experiments, and form stable native structures under slightly favourable thermodynamic conditions [ , ]. The ability of IDPs to be both partially disordered and poised to fold up with slight thermodynamic input seems to be a simple manifestation of their one-state downhill folding character [ ]. Such folding characteristics enable their operation as conformational rheostats, that is molecular devices capable of producing analogical signals in response to binary stimuli such as binding to specific partners [ , , ] Figure 7 , left.
In this light, the functional complexity and multiple binding modes reported on IDPs could be explained as emerging from the coupling between binding and downhill folding. There is mounting evidence that the complex binding modes observed on IDPs involve gradual conformational changes rather than binary transitions. The partially folded conformational ensemble of this domain can gradually become more or less structured by coupling folding to a signal such as binding to one or various partners.
For example, NCBD nuclear co-activator-binding domain has been classified as an IDP [ , ] that binds multiple partners by folding into different conformers [ , , ]. At the same time, NCBD is capable of folding into a three-helix bundle structure in the presence of stabilizing agents, and it does so following a gradual process one-state downhill according to the multivariate analysis of multi-probe experimental data and computer simulations [ ].
Another interesting case is the PSBDs peripheral subunit-binding domains from several multienzymatic complexes, such as the pyruvate and 2-oxoglutarate dehydrogenases [ ], which include the first identified examples of one-state downhill folding [ ]. In these multienzymes, the catalytic process involves four steps catalysed by three subunits E1, E2 and E3 that form a dynamic macromolecular complex, one that is fully controlled by the interactions between the PSBD from the E2 subunit and the E1 and E3 subunits [ ]. Certain DNA-binding proteins, such as homeodomains, are also likely candidates for conformational rheostats.
These domains face the enormous challenge of finding a short target sequence within the enormous pool of potential binding sites provided by genomic DNA. DNA sliding has been studied theoretically [ ], computationally [ ] and experimentally using single-molecule methods [ ] and paramagnetic relaxation enhancement NMR [ ]. The molecular mechanism by which DNA-binding proteins implement these two binding modes remains largely unclear, however.
But we now know that DNA-binding domains are marginally co-operative fast folders [ ], exhibit partial disorder when unbound to DNA [ ], and seem to fold via a downhill mechanism [ ]. These properties suggest a molecular rheostat in which the conformational motions of a partially unfolded domain are exploited to counterbalance DNA processivity and sliding speed during non-specific binding, and ensure quick locking into the target sequence.
These domains also bind non-specifically to DNA in a manner that seems to be DNA-sequence-dependent [ ], which further suggests a homing-to-target mechanism mediated by conformational selection. Finally, conformational rheostats also offer very attractive possibilities for technological applications. A first effort in this direction has targeted the design of high-performance biosensors based on gradual conformational changes coupled to proton binding [ ].
The authors of that work exploited the natural properties of the BBL domain in terms of folding [ ] and proton binding [ ] to engineer a pH ionic strength sensor with linear response over five orders of magnitude in analyte concentration, instead of the two orders that are inherent to conformational switches Figure 7 , right. Moreover, these sensors exhibited ultrafast response thanks to the microsecond folding kinetics of BBL and the gradual coupling between folding and binding [ ].
This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution Licence 4. We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address. Skip to main content. Abstract Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways.
Figure 1 Protein folding inside the cell A new protein is synthetized at the ribosome as determined by the activation of gene expression. Ultrafast kinetic techniques Kinetic experiments measure the conformational relaxation of the protein in response to a perturbation, and have time resolution determined by how quickly the perturbation is enacted Figure 2. Figure 2 Experimental and computational approaches for investigating fast protein folding Ultrafast kinetic perturbation methods left: Single-molecule spectroscopy Single-molecule methods can resolve the stochastic conformational fluctuations of the protein in equilibrium conditions, and thus do not need a fast perturbation.
Probing fast folding at atomic resolution NMR nuclear magnetic resonance is particularly attractive for protein folding studies because it provides both atomic-resolution and dynamic information [ 50 ]. Atomistic computer simulations MD molecular dynamics simulations offer atomistic structural resolution and dynamic information with virtually infinite time resolution.
Figure 3 Roadmap of folding timescales The chart shows the timescales associated with the various structural events that take place in protein folding reactions and the experimental and computational methods that are used to probe them.
Hydrophobic collapse The random collapse of the unfolded polypeptide chain to exclude hydrophobic side chains from surrounding solvent is one of the key events that take place during folding. Loop formation The closure of loops determines the formation of interactions between secondary-structure elements to form supersecondary and tertiary arrangements.
Secondary structure Investigating the timescales for secondary-structure elements required combining the fastest kinetic methods with small peptides that were able to form stable secondary structures on their own. Topological reorganization The rearrangement of secondary-structure elements to form native tertiary interactions on a randomly collapsed globule should occur intrinsically more slowly than the motions described before because it requires breaking pre-formed interactions before the protein can reconfigure.
FAST PROTEIN FOLDING Understanding the determinants of protein folding rates Figure 3 provides an entry point to investigate the determinants of the over six orders of magnitude spread in folding rates that is observed in natural single-domain proteins [ ], and, as an ancillary issue, to estimate the speed limit for protein folding reactions [ ].
For these purposes, we can utilize a simple folding rate expression derived from the energy landscape approach [ 6 ]: Figure 4 The determinants of protein folding rates Left: Searching for microsecond folding proteins New kinetic techniques also opened the opportunity to resolve the folding—unfolding kinetics of proteins that were too fast for the stopped-flow method. Downhill folding Another exciting implication of fast folding research is the downhill folding scenario.
Thermodynamic folding barriers from calorimetry DSC differential scanning calorimetry is extremely sensitive to the conformational heterogeneity of protein folding reactions [ ]. Reconstructing folding landscapes from multi-probe unfolding experiments Fast folding proteins exhibit marginal unfolding co-operativity that results in non-concerted structural disassembly [ ]. Folding interaction networks at atomic resolution The non-concerted unfolding behaviour of fast-folding proteins can be taken one step further to determine the network of interactions that stabilize the native structure [ ].
Structural analysis of excited states in protein folding RD-NMR see above has been widely used to detect minimally populated species associated with protein conformational changes taking place during catalysis, ligand binding or DNA sliding motions, which tend to occur in the sub-millisecond to millisecond timescale [ 54 ].
Folding pathways and mechanisms In addition to obtaining structural information, it is also important to measure dynamic events such as folding transitions and microscopic pathways because they reveal the heterogeneity of mechanisms. Figure 5 Transition paths in protein folding The folding free energy landscape of a single-domain protein is often represented with a simple 1D surface with two minima for the native N and unfolded states U , and a more or less pronounced barrier separating them left.
Figure 7 Biological and technological roles of conformational rheostats Left: R81 — R91 , doi: We review progress on these problems. In a few cases, computer simulations of the physical forces in chemically detailed models have now achieved the accurate folding of small proteins. We have learned that proteins fold rapidly because random thermal motions cause conformational changes leading energetically downhill toward the native structure, a principle that is captured in funnel-shaped energy landscapes. And thanks in part to the large Protein Data Bank of known structures, predicting protein structures is now far more successful than was thought possible in the early days.
What began as three questions of basic science one half-century ago has now grown into the full-fledged research field of protein physical science.
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