Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Pashley, Clare L.; Morgan, Gareth J.; Kalverda, Arnout P.; Thompson, Gary S.; Kleanthous, Colin; Radford, Sheena E.

doi:10.1016/j.jmb.2011.12.041

Cited by 29 publications

(57 citation statements)

References 87 publications

(166 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The biophysically amenable nature of the recently discovered chaperone Spy and one of its in vivo clients, Im7, together with the previously established mechanism of Im7 folding (Capaldi et al, 2001, 2002; Friel et al, 2009; Gsponer et al, 2006; Pashley et al, 2012; Whittaker et al, 2007), afforded us the opportunity to analyze the forces governing how an unfolded protein binds, folds, and is subsequently released by a chaperone.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, conditions and mutants of Im7 have been developed that enable both partially and fully unfolded Im7 variants to remain soluble (Capaldi et al, 2001, 2002; Friel et al, 2009; Gsponer et al, 2006; Pashley et al, 2012; Whittaker et al, 2007). These Im7 variants therefore avoid aggregation, one of the major challenges in working with chaperone clients and a key reason that the number of kinetic and thermodynamic studies on chaperones is limited.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Forces Driving Chaperone Action

Koldewey

Stull

Horowitz

et al. 2016

Cell

View full text Add to dashboard Cite

SUMMARY It is still unclear what molecular forces drive chaperone-mediated protein folding. Here, we obtain a detailed mechanistic understanding of the forces that dictate the four key steps of chaperone-client interaction: initial binding, complex stabilization, folding, and release. Contrary to the common belief that chaperones recognize unfolding intermediates by their hydrophobic nature, we discover that the model chaperone Spy uses long-range electrostatic interactions to rapidly bind to its unfolded client protein Im7. Short-range hydrophobic interactions follow, which serve to stabilize the complex. Hydrophobic collapse of the client protein then drives its folding. By burying hydrophobic residues in its core, the client’s affinity to Spy decreases, which causes client release. By allowing the client to fold itself, Spy circumvents the need for client-specific folding instructions. This mechanism might help explain how chaperones can facilitate the folding of various unrelated proteins.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Forces Driving Chaperone Action

Koldewey

Stull

Horowitz

et al. 2016

Cell

View full text Add to dashboard Cite

show abstract

“…There is growing evidence that the DSE can be compact under native conditions and can form native and nonnative clusters of hydrophobic residues, native and nonnative secondary structure, and even nonnative electrostatic interactions, thus the NTL9 is unlikely to be a special case (10,12,14,18,22,27,46,47). NTL9 is emerging as a popular system for long molecular dynamics simulations and the data presented here will help provide a rigorous benchmark for these efforts (45,(48)(49)(50).…”

Section: Discussionmentioning

confidence: 97%

“…protein folding | protein stability | ϕ-value analysis | double-mutant cycle T he properties of nonnative states of proteins have attracted considerable attention because they can play critical roles in cell signaling, translocation across membranes, temperaturesensitive phenotypes, and in a wide range of protein deposition diseases (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The properties of unfolded states are also relevant to any discussion of intrinsically disordered proteins (IDPs) (1).…”

mentioning

confidence: 99%

Energetically significant networks of coupled interactions within an unfolded protein

Cho

Meng

Sato

et al. 2014

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

View full text Add to dashboard Cite

Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pK a measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.protein folding | protein stability | ϕ-value analysis | double-mutant cycle T he properties of nonnative states of proteins have attracted considerable attention because they can play critical roles in cell signaling, translocation across membranes, temperaturesensitive phenotypes, and in a wide range of protein deposition diseases (1-14). The properties of unfolded states are also relevant to any discussion of intrinsically disordered proteins (IDPs) (1). The denatured state ensemble (DSE) can be populated under strongly denaturing conditions and studied at equilibrium; however, the physiologically relevant DSE is the state that is in equilibrium with the folded state under native conditions. This state is difficult to study directly and most studies of the DSE under native conditions have made use of indirect approaches. Nevertheless, experimental and theoretical work has shown that the DSE populated under physiological conditions is not a random coil, but can contain native and nonnative structure, stabilized by local and nonlocal interactions. However, very little is known about the energetics of these interactions, particularly ones involving residues distant in sequence, or about energetically coupled interactions involving multiple residues in the . These are a critical piece of the folding puzzle; they impact protein aggregation and folding, and have important implications for the properties of IDPs. Recent studies offer evidence for hydrophobic clusters, electrostatic interactio...

show abstract

“…The FRET experiments showed that the dimensions of the ensemble of collapsed protein molecules are smaller than those of the ensemble of unfolded molecules but larger than those of the folded state [99]. The collapsed state was described as a globular state of proteins that is akin to the collapsed state of polymers, as it is predominantly disordered (reviewed by Haran [9] and Udgaonkar [99,[167][168][169]). …”

Section: Multiprobe Fret Studies Of the Initial Collapse Transitionmentioning

confidence: 99%

Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

Orevi

Rahamim

Shemesh

et al. 2014

Bio-Algorithms and Med-Systems

View full text Add to dashboard Cite

The protein folding problem would be considered "solved" when it will be possible to "read genes", i.e., to predict the native fold of proteins, their dynamics, and the mechanism of fast folding based solely on sequence data. The long-term goal should be the creation of an algorithm that would simulate the stepwise mechanism of folding, which constrains the conformational space and in which random search for stable interactions is possible. Here, we focus attention on the initial phases of the folding transition starting with the compact disordered collapsed ensemble, in search of the initial sub-domain structural biases that direct the otherwise stochastic dynamics of the backbone. Our studies are designed to test the "loop hypothesis", which suggests that fast closure of long loop structures by non-local interactions between clusters of mainly non-polar residues is an essential conformational step at the initiation of the folding transition of globular proteins. We developed and applied experimental methods based on time-resolved resonance excitation energy transfer (trFRET) measurements combined with fast mixing methods and studied the initial phases of the folding of Escherichia coli adenylate kinase (AK). A series of AK mutants were prepared, in which the ends of selected backbone segments that form long closed loops or secondary structure elements were labeled by donors and acceptors of excitation energy. The end-to-end distance distributions of such segments were determined under equilibrium and during the fast folding transitions. These experiments show that three out of seven long loops that were labeled in the AK molecule are closed very early in the transition. The N terminal 26-residue loop (loop I) is closed in < 200 μs after the initiation of folding, while the β strand included in loop I is still disordered. The closure of the second 44-residue loop (loop II, which starts at the end of loop I) is also complete within < 300 μs. Four other loops as well as five secondary structures of the CORE domain of AK (an α helix and four β strands) are formed at a late step, at a rate of 0.5 ± 0.3 s -1 , the rate of the cooperative folding of the molecule. These experiments reveal a hierarchically ordered pathway of folding of the AK molecule, ranging from microseconds to seconds. The results reviewed here, obtained mainly from studying a small number of model proteins, support the counterintuitive mechanism whereby non-local interactions are effective in the initiation of the folding pathways. The experiments presented demonstrate the importance of mapping the rates of sub-domain structural transitions along the folding transition, in situ, in the context of the other sections of the chain, whether folded or disordered. These experiments also show the power of the time-resolved FRET measurements in achieving this goal. A large body of data obtained by theoretical and experimental studies that support, or can accommodate, the loop hypothesis is reviewed. We suggest that mapping multiple sub-domain structural tr...

show abstract

Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

Cited by 29 publications

References 87 publications

Forces Driving Chaperone Action

Forces Driving Chaperone Action

Energetically significant networks of coupled interactions within an unfolded protein

Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

Contact Info

Product

Resources

About