The transition state for coupled folding and binding of a disordered DNA binding domain resembles the unbound state.

The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable α-helices upon target DNA binding. Here, we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state.

Binding and folding in transcriptional complexes.

Transcription factors are among the classes of proteins with the highest levels of disorder. Investigation of these regulatory proteins is uncovering not just the mechanisms that underlie gene regulation, but relationships that apply to all intrinsically disordered proteins. Recent studies confirm that binding does not necessarily induce folding but that when it does, it tends to follow induced fit mechanisms.

Charge Interactions Modulate the Encounter Complex Ensemble of Two Differently Charged Disordered Protein Partners of KIX.

Disordered proteins play important roles in cell signaling and are frequently involved in protein-protein interactions. They also have a larger proportion of charged and polar residues than their folded counterparts. Here, we developed a structure-based model and applied molecular dynamics simulations to examine the presence and importance of electrostatic interactions in the binding processes of two differently charged intrinsically disordered ligands of the KIX domain of CBP.

Stopped-Flow Kinetic Techniques for Studying Binding Reactions of Intrinsically Disordered Proteins.

Intrinsically disordered proteins are abundant in signaling processes such as transcription. Suitable binding and unbinding rates of proteins with their partners are critical for allowing them to perform their biological roles. Understanding how these are achieved, and indeed designing strategies for intervening or modulating related biological processes, therefore requires kinetic studies.

Two Differential Binding Mechanisms of FG-Nucleoporins and Nuclear Transport Receptors.

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an "archetypal-fuzzy" complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1.

Hsp70 Inhibits the Nucleation and Elongation of Tau and Sequesters Tau Aggregates with High Affinity.

As a key player of the protein quality control network of the cell, the molecular chaperone Hsp70 inhibits the aggregation of the amyloid protein tau. To date, the mechanism of this inhibition and the tau species targeted by Hsp70 remain unknown. This is partly due to the inherent difficulty of studying amyloid aggregates because of their heterogeneous and transient nature.

pKID Binds to KIX via an Unstructured Transition State with Nonnative Interactions.

Understanding the detailed mechanism of interaction of intrinsically disordered proteins with their partners is crucial to comprehend their functions in signaling and transcription. Through its interaction with KIX, the disordered pKID region of CREB protein is central in the transcription of cAMP responsive genes, including those involved in long-term memory. Numerous simulation studies have investigated these interactions.

Phosphorylation of the IDP KID Modulates Affinity for KIX by Increasing the Lifetime of the Complex.

Intrinsically disordered proteins (IDPs) are known to undergo a range of posttranslational modifications, but by what mechanism do such modifications affect the binding of an IDP to its partner protein? We investigate this question using one such IDP, the kinase inducible domain (KID) of the transcription factor CREB, which interacts with the KIX domain of CREB-binding protein upon phosphorylation. As with many other IDPs, KID undergoes coupled folding and binding to form α-helical structure upon interacting with KIX. This single site phosphorylation plays an important role in the control of transcriptional activation in vivo.

Affinity of IDPs to their targets is modulated by ion-specific changes in kinetics and residual structure.

Intrinsically disordered proteins (IDPs) are characterized by a lack of defined structure. Instead, they populate ensembles of rapidly interconverting conformations with marginal structural stabilities. Changes in solution conditions such as temperature and crowding agents consequently affect IDPs more than their folded counterparts.

Conserved Helix-Flanking Prolines Modulate Intrinsically Disordered Protein:Target Affinity by Altering the Lifetime of the Bound Complex.

Appropriate integration of cellular signals requires a delicate balance of ligand-target binding affinities. Increasing the level of residual structure in intrinsically disordered proteins (IDPs), which are overrepresented in these cellular processes, has been shown previously to enhance binding affinities and alter cellular function. Conserved proline residues are commonly found flanking regions of IDPs that become helical upon interacting with a partner protein.

Role of non-native electrostatic interactions in the coupled folding and binding of PUMA with Mcl-1.

PUMA, which belongs to the BH3-only protein family, is an intrinsically disordered protein (IDP). It binds to its cellular partner Mcl-1 through its BH3 motif, which folds upon binding into an α helix. We have applied a structure-based coarse-grained model, with an explicit Debye-Hückel charge model, to probe the importance of electrostatic interactions both in the early and the later stages of this model coupled folding and binding process.

Mechanistic roles of protein disorder within transcription.

Understanding the interactions of proteins involved in transcriptional regulation is critical to describing biological systems because they control the expression profile of the cell. Yet sadly they belong to a less well biophysically characterized subset of proteins; they frequently contain long disordered regions that are highly dynamic. A key question therefore is, why? What functional roles does protein disorder play in transcriptional regulation? Experimental data exemplifying these roles are starting to emerge, with common themes being enabling complexity within networks and quick responses.

GADIS: Algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins.

Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs.

Insights into Coupled Folding and Binding Mechanisms from Kinetic Studies.

Intrinsically disordered proteins (IDPs) are characterized by a lack of persistent structure. Since their identification more than a decade ago, many questions regarding their functional relevance and interaction mechanisms remain unanswered. Although most experiments have taken equilibrium and structural perspectives, fewer studies have investigated the kinetics of their interactions.

Transient misfolding dominates multidomain protein folding.

Neighbouring domains of multidomain proteins with homologous tandem repeats have divergent sequences, probably as a result of evolutionary pressure to avoid misfolding and aggregation, particularly at the high cellular protein concentrations. Here we combine microfluidic-mixing single-molecule kinetics, ensemble experiments and molecular simulations to investigate how misfolding between the immunoglobulin-like domains of titin is prevented. Surprisingly, we find that during refolding of tandem repeats, independent of sequence identity, more than half of all molecules transiently form a wide range of misfolded conformations.

Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state.

A mechanistic model of tau amyloid aggregation based on direct observation of oligomers.

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L.

Interplay between partner and ligand facilitates the folding and binding of an intrinsically disordered protein.

Protein-protein interactions are at the heart of regulatory and signaling processes in the cell. In many interactions, one or both proteins are disordered before association. However, this disorder in the unbound state does not prevent many of these proteins folding to a well-defined, ordered structure in the bound state.

Allostery within a transcription coactivator is predominantly mediated through dissociation rate constants.

The kinase-inducible domain interacting (KIX) domain of CREB binding protein binds to multiple intrinsically disordered transcription factors in vivo at two distinct sites on its surface. Several reports have been made of allosteric communication between these two sites in this well-characterized model system. In this work, we have performed fluorescence stopped-flow measurements to investigate the kinetics of binding of five KIX binding proteins.

Mechanism of assembly of the non-covalent spectrin tetramerization domain from intrinsically disordered partners.

Interdomain interactions of spectrin are critical for maintenance of the erythrocyte cytoskeleton. In particular, "head-to-head" dimerization occurs when the intrinsically disordered C-terminal tail of β-spectrin binds the N-terminal tail of α-spectrin, folding to form the "spectrin tetramer domain". This non-covalent three-helix bundle domain is homologous in structure and sequence to previously studied spectrin domains.

Remarkably fast coupled folding and binding of the intrinsically disordered transactivation domain of cMyb to CBP KIX.

Association rates for interactions between folded proteins have been investigated extensively, allowing the development of computational and theoretical prediction methods. Less is known about association rates for complexes where one or more partner is initially disordered, despite much speculation about how they may compare to those for folded proteins. We have attached a fluorophore to the N-terminus of the 25 amino acid cMyb peptide used previously in NMR and equilibrium studies (termed FITC-cMyb), and used this to monitor the kinetics of its interaction with the KIX protein.

Protein folding: adding a nucleus to guide helix docking reduces landscape roughness.

The elongated three-helix-bundle spectrin domains R16 and R17 fold and unfold unusually slowly over a rough energy landscape, in contrast to the homologue R15, which folds fast over a much smoother, more typical landscape. R15 folds via a nucleation-condensation mechanism that guides the docking of the A and C-helices. However, in R16 and R17, the secondary structure forms first and the two helices must then dock in the correct register.

Separating the effects of internal friction and transition state energy to explain the slow, frustrated folding of spectrin domains.

The elongated three-helix bundle domains spectrin R16 and R17 fold some two to three orders of magnitude more slowly than their homologue R15. We have shown that this slow folding is due, at least in part, to roughness in the free-energy landscape of R16 and R17. We have proposed that this roughness is due to a frustrated search for the correct docking of partly preformed helices.

Binding of the molecular chaperone αB-crystallin to Aβ amyloid fibrils inhibits fibril elongation.

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ(42) fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ(42).