Criteria for downhill protein folding: Calorimetry, chevron plot, kinetic relaxation, and single‐molecule radius of gyration in chain models with subdued degrees of cooperativity

116

169

We have investigated the site-specific folding kinetics of a photoswitchable cross-linked ␣-helical peptide by using single 13 C ‫؍‬ 18 O isotope labeling together with time-resolved IR spectroscopy. We observe that the folding times differ from site to site by a factor of eight at low temperatures (6°C), whereas at high temperatures (45°C), the spread is considerably smaller. The trivial sum of the site signals coincides with the overall folding signal of the unlabeled peptide, and different sites fold in a noncooperative manner. Moreover, one of the sites exhibits a decrease of hydrogen bonding upon folding, implying that the unfolded state at low temperature is not unstructured. Molecular dynamics simulations at low temperature reveal a stretched-exponential behavior which originates from parallel folding routes that start from a kinetically partitioned unfolded ensemble. Different metastable structures (i.e., traps) in the unfolded ensemble have a different ratio of loop and helical content. Control simulations of the peptide at high temperature, as well as without the cross-linker at low temperature, show faster and simpler (i.e., single-exponential) folding kinetics. The experimental and simulation results together provide strong evidence that the rate-limiting step in formation of a structurally constrained ␣-helix is the escape from heterogeneous traps rather than the nucleation rate. This conclusion has important implications for an ␣-helical segment within a protein, rather than an isolated ␣-helix, because the cross-linker is a structural constraint similar to those present during the folding of a globular protein.cooperativity ͉ infrared spectroscopy ͉ molecular dynamics simulation ͉ peptide folding

Section: Resultsmentioning

confidence: 94%

α-Helix folding in the presence of structural constraints

Ihalainen¹,

Paoli

Muff

et al. 2008

116

169

“…Thus, it has become increasingly clear that biology has made use of the manifold polymeric states available to a polypeptide chain (18). Even for proteins that can be folded, in view of recent advances in the study of possible noncooperative ''downhill'' folding (23)(24)(25), the traditional notion of cooperative folding as a functional requirement for globular proteins seems an oversimplification (26). For many tasks involved in cellular function, intrinsically disordered proteins offer several unique attributes.…”

mentioning

confidence: 99%

Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity

Borg

Mittag²,

Pawson³

et al. 2007

Self Cite

215

262

Regulation of biological processes often involves phosphorylation of intrinsically disordered protein regions, thereby modulating protein interactions. Initiation of DNA replication in yeast requires elimination of the cyclin-dependent kinase inhibitor Sic1 via the SCF Cdc4 ubiquitin ligase. Intriguingly, the substrate adapter subunit Cdc4 binds to Sic1 only after phosphorylation of a minimum of any six of the nine cyclin-dependent kinase sites on Sic1. To investigate the physical basis of this ultrasensitive interaction, we consider a mean-field statistical mechanical model for the electrostatic interactions between a single receptor site and a conformationally disordered polyvalent ligand. The formulation treats phosphorylation sites as negative contributions to the total charge of the ligand and addresses its interplay with the strength of the favorable ligand-receptor contact. Our model predicts a threshold number of phosphorylation sites for receptorligand binding, suggesting that ultrasensitivity in the Sic1-Cdc4 system may be driven at least in part by cumulative electrostatic interactions. This hypothesis is supported by experimental affinities of Cdc4 for Sic1 fragments with different total charges. Thus, polyelectrostatic interactions may provide a simple yet powerful framework for understanding the modulation of protein interactions by multiple phosphorylation sites in disordered protein regions.cooperativity ͉ cyclin-dependent kinase inhibitor ͉ DNA replication ͉ phosphorylation ͉ yeast cell cycle

“…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). 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)(15)(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

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

Huang

Ying

Fersht

2009

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...