Forced Folding of a Disordered Protein Accesses an Alternative Folding Landscape

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand‐specific functional structures. For such multi‐structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single‐molecule and ensemble techniques to compare ligand‐induced and osmolyte‐forced folding of α‐synuclein. Our results reveal context‐dependent modulation of the protein′s folding landscape, suggesting that the codes for the protein′s native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins.     

Folding versus self-assembling

Model foldable polymers with sequences of rigid hydrophobic chromophores and flexible hydrophilic tetra(ethylene glycol) were synthesized and used as a paradigm for studying molecular-folding and self-assembly phenomena. Our results demonstrate that intramolecular association or folding prevails over intermolecular interaction or self-assembling in the concentration region from 1 microM to 0.1 M. Importantly, folded polymeric nanostructures have absorption and fluorescence properties that are distinct from those of unfolded polymers or free monomers. We hypothesize that the origins of folding and self-assembly come from interactions between molecular units, and that the key parameter that regulates the on-and-off of such interactions is the distance R separating the two molecular units. Each molecular unit produces a characteristic force field, and when another molecular unit enters this field, the probability that the two units will interact increases significantly. A preliminary estimate of the radius of such a force field for the perylene tetracarboxylic diimide chromophore is about 90-120 A. As a result, phenomena associated with folding or self-assembly of molecular species are observed when these conditions are met in solution.     

Folding propensity of cyclohexylether-delta-peptides

[structure: see text] Linear (n = 2-18) and cyclic oligomers (n = 3-8) of a cyclohexylether-delta-amino acid (COA) were prepared in high yield and stereopurity. CD spectra of the linear oligomers were indicative of secondary structure formation. X-ray crystal structures of cyclic COA oligomers revealed hydrophobic packing and internal 5- and 10-membered-ring hydrogen bonds. Ether and amide oxygens reside preferably in an ap orientation. This conformational locking is apparently broken by a C-2 substituent in an asymmetric cyclotrimer, for which a zeolithe-like tubular structure was found.     

Folding of small origamis

A model that preserves the known thermodynamic properties of double stranded DNA is introduced to study the formation of more complex DNA constructions, such as small origamis or Holliday junctions. We show that the thermodynamic behaviour of these complex DNA constructions is not only given by their sequence but also by their topology.     

Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme

UDP‐glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re‐glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re‐glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.     

Folding kinetics of a polymer

We present the results of computer simulations giving a kinetic insight into the liquid-to-solid transition of a homopolymer chain with short-range interactions. By calculating the absolute rates in each direction of the transition, using molecular dynamics employing the forward flux sampling scheme, we provide the phase diagram based on purely kinetic data, and compare it with the results from Monte Carlo simulations. Additionally, we present and discuss a remarkably simple and general relation between the polymer topology and the folding pathway, and show that the eigenvalue spectrum of a matrix defined by non-bonded contacts (the Laplacian matrix) provides an insight into the nonequilibrium ensembles of these trajectories. In particular, the Laplacian matrix seems to identify a large fraction of configurations on the folding pathway at the free energy maximum that have a very low probability of reaching the crystallized state. This implies that the eigenvalues of this matrix may be suitable additional reaction coordinates to describe the folding transition of chain molecules.     

Folding of alternating dialkylsilylene-spaced donor-acceptor copolymers: the oligomer approach

A series of oligmers with donor-acceptor pairs separated by diisopropylsilylene (iPr(2)Si) spacers, composed of monomer 4b, dimer 5, trimer 6, and tetramer 7, were synthesized to scrutinize the folding behavior. Monomer 4a with a dimethylsilylene (Me(2)Si) spacer was also prepared for comparison. The 4-aminostyrene moiety was used as the donor and the stilbene moiety as the acceptor. Both steady-state and time-resolved fluorescence spectroscopic measurement were made. Regardless of the substituents on the silicon atom, the emission spectra of 4a and 4b exhibit both local excited (LE) emission of the acceptor chromophore and emission from the charge-separated state (CT emission), which are similar to that of the corresponding Me(2)Si-spaced copolymer 2a with the same donor and acceptor chromophores, but different from that of the copolymer with the iPr(2)Si spacer 2b. Dimer 5 behaves like 4 and 2a. As the chain length of the oligomers increases, the emission properties of the higher homologues become prone to that of 2b. Thus, tetramer 7 exhibits emission from the charge-transfer complex, which is essentially same as that of 2b. Moreover, charge-transfer absorptions emerge in 6 and 7. These results suggest that the folding nature of oligomers approaches that of the corresponding polymer, as the degree of oligomerization increases, and the electronic interactions between adjacent donor-acceptor pairs are controlled by the steric effect of the substituents on the silicon atoms and concomitant amplification of the stabilizing energy by extending the distance of the folding structure.     

Folding a conjugated chain: oligo(o-phenyleneethynylene-alt-p-phenyleneethynylene)

An oligo( o-phenyleneethynylene- alt- p-phenyleneethynylene) was synthesized to create a conjugated molecule capable of adopting a helical secondary structure. A special feature of such a folded molecule is that the effective directions of energy/charge transport via covalent conjugation and through pi-pi stacking are converged to be along the helix axis. The transition from random conformations to the helix, driven by solvophobic and aromatic stacking interactions, was controlled by solvent properties. UV-vis and fluorescence spectroscopies gave supportive evidence for the folding process.     

Protein Folding: A Perspective from Theory and Experiment

The mechanism of protein folding (represented schematically below) is one of the most fascinating problems in the field of chemical reactions. This review presents the progess made recently in understanding key elements of this reaction and describes a solution to the often quoted Levinthal Paradox.     

Folding structures of isolated peptides as revealed by gas-phase mid-infrared spectroscopy.

To understand the intrinsic properties of peptides, which are determined by factors such as intramolecular hydrogen bonding, van der Waals bonding and electrostatic interactions, the conformational landscape of isolated protein building blocks in the gas phase was investigated. Here, we present IR-UV double-resonance spectra of jet-cooled, uncapped peptides containing a tryptophan (Trp) UV chromophore in the 1000-2000 cm(-1) spectral range. In the series Trp, Trp-Gly and Trp-Gly-Gly (where Gly stands for glycine), the number of detected conformers was found to decrease from six (Snoek et al., PCCP, 2001, 3, 1819) to four and two, respectively, which indicates a trend to relaxation to a global minimum. Density functional theory calculations reveal that the O-H in-plane bending vibration, together with the N-H in-plane bend ing and the peptide C=O stretching vibrations, is a sensitive probe to hydrogen bonding and, thus, to the folding of the peptide backbone in these structures. This enables the identification of spectroscopic fingerprints for the various conformational structures. By comparing the experimentally observed IR spectra with the calculated spectra, a unique conformational assignment can be made in most cases. The IR-UV spectrum of a Trp-containing nonapeptide (Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu) was recorded as well and, although the IR spectrum is less well-resolved (and it probably results from different isomers), groups of amide I (peptide C=O stretching) and amide II (N-H in-plane bending) bands can still be recognised, in agreement with predictions at the AM1 level.     

Folding Behavior of Polypeptides. A Monte Carlo Study of Simplified Models

Summary. A simple model of polypeptide chains was designed and studied. The chains were constructed on a flexible [310] lattice and consisted of united atoms located at the position of alpha carbons. Each united atom represented amino acid residues of two kinds: hydrophilic and hydrophobic. The sequence of the residues was assumed to be characteristic for α- and β-type of proteins. The force field used consisted of the long-range contact potential between polymer segments, the short range repulsion, and the local potential preferring conformational states characteristic for α-helices and β-strands. The Monte Carlo simulations of this model were carried out using the replica exchange technique coupled with the histogram method. The influence of temperature and the local potential on the size and internal structure of collapsed low temperature chains were studied. Thermodynamics of these systems consisting mainly of α and β secondary structures were determined. The properties of the coil-to-globule transition were presented and compared with other theoretical predictions and simulation results.     

Folding Behavior of Polypeptides. A Monte Carlo Study of Simplified Models

A simple model of polypeptide chains was designed and studied. The chains were constructed on a flexible [310] lattice and consisted of united atoms located at the position of alpha carbons. Each united atom represented amino acid residues of two kinds: hydrophilic and hydrophobic. The sequence of the residues was assumed to be characteristic for α- and β-type of proteins. The force field used consisted of the long-range contact potential between polymer segments, the short range repulsion, and the local potential preferring conformational states characteristic for α-helices and β-strands. The Monte Carlo simulations of this model were carried out using the replica exchange technique coupled with the histogram method. The influence of temperature and the local potential on the size and internal structure of collapsed low temperature chains were studied. Thermodynamics of these systems consisting mainly of α and β secondary structures were determined. The properties of the coil-to-globule transition were presented and compared with other theoretical predictions and simulation results.     

Folding degrees of azurins and pseudoazurins. Implications for structure and function

A quantitative measure of the degree of folding of azurins and pseudoazurins has been made. We have found that the reduction potential of azurins and pseudoazurins is a function of the contribution to the degree of folding of His117, a key amino acid in electron transfer which is directly bonded to copper in these proteins. The folding degree of His117 explains 95% of the variance in the experimental values of the reduction potential of azurins and pseudoazurins. The change in the folding degree of this amino acid influences several geometric parameters of the main backbones of these proteins. Among them, the angle formed between N(His117)...Cu...S(Cys112), which plays an important role in electron transport, but not the N(His117)...Cu distance, shows some non-linear correlation with the reduction potential of azurins and pseudoazurins. However, it is only able to explain less than 75% in the variance of the reduction potential of these proteins instead of the 95% explained by the folding degree of His117.     

高分子的折叠与聚集

总结了作者有关高分子折叠和聚集方面的工作。最初,作者研究了聚（N-异丙基丙烯酰胺）（PNIPAM）均聚物的折叠或叫做“线团-塌缩球的转变”,然后研究了含有疏水和亲水基团的PNIPAM共聚物的折叠。作者研究了疏水作用和亲水作用对折叠的影响,发现了融化球,有序线团等折叠过程中的中间态。另一方面,作者研究了两亲性高分子在水中的聚集与稳定。作者的结果表明,如果高分子链所形成的稳定聚集体为核-壳结构,则每个亲水基团所占有的面积为一个常数。如聚集体不是核-壳结构,即部分亲水基团分布在聚集体内部,则上述关系不再成立。随亲水基团含量的增加,聚集体将由球状变为超枝化结构。     

Catalyst-Controlled Transannular Polyketide Cyclization Cascades: Selective Folding of Macrocyclic Polyketides

The biomimetic synthesis of aromatic polyketides from macrocyclic substrates by means of catalyst-controlled transannular cyclization cascades is described. The macrocyclic substrates, which feature increased stability and fewer conformational states, were thereby transformed into several distinct polyketide scaffolds. The catalyst-controlled transannular cyclizations selectively led to aromatic polyketides with a defined folding and oxygenation pattern, thus emulating β-keto-processing steps of polyketide biosynthesis.     

Metalloporphyrin Dendrimers with Folding Arms

Rigid and flexible linkers are combined in the new dendrimer shown schematically in the picture, which contains nine metalloporphyrin units (Porph). The construction is such that the four “arms” of the dendrimer can fold in a cooperative and predetermined manner in response to added 1,4-diazabicyclo[2.2.2]octane (DABCO).     

Folding funnels: the key to robust protein structure prediction

Natural proteins fold because their free energy landscapes are funneled to their native states. The degree to which a model energy function for protein structure prediction can avoid the multiple minima problem and reliably yield at least low-resolution predictions is also dependent on the topography of the energy landscape. We show that the degree of funneling can be quantitatively expressed in terms of a few averaged properties of the landscape. This allows us to optimize simplified energy functions for protein structure prediction even in the absence of homology information. Here we outline the optimization procedure in the context of associative memory energy functions originally introduced for tertiary structure recognition and demonstrate that even partially funneled landscapes lead to qualitatively correct, low-resolution predictions.