Trp Side Chain Research Articles

Integrating 19F Distance Restraints for Accurate Protein Structure Determination by Magic Angle Spinning NMR Spectroscopy.

Traditional protein structure determination by magic angle spinning (MAS) solid-state NMR spectroscopy primarily relies on interatomic distances up to 8 Å, extracted from 13C-, 15N-, and 1H-based dipolar-based correlation experiments. Here, we show that 19F fast (60 kHz) MAS NMR spectroscopy can supply additional, longer distances. Using 4F-Trp,U-13C,15N crystalline Oscillatoria agardhii agglutinin (OAA), we demonstrate that judiciously designed 2D and 3D 19F-based dipolar correlation experiments such as (H)CF, (H)CHF, and FF can yield interatomic distances in the 8-16 Å range. Incorporation of fluorine-based restraints into structure calculation improved the precision of Trp side chain conformations as well as regions in the protein around the fluorine containing residues, with notable improvements observed for residues in proximity to the Trp pairs (W10/W17 and W77/W84) in the carbohydrate-binding loops, which lacked sufficient long-range 13C-13C distance restraints. Our work highlights the use of fluorine and 19F fast MAS NMR spectroscopy as a powerful structural biology tool.

Interactions of Li+ ions with NCS1: A potential mechanism of Li+ neuroprotective action against psychotic disorders

Li+ based drugs have been used for the treatment of psychiatric disorders due to their mood stabilizing role for decades. Recently, several studies reported the protective effect of Li+ against severe neuropathologies such as Parkinson's, Alzheimer's, and Huntington's disease. Surprisingly, despite a broad range of Li+ effects on neurological conditions, little is known about its molecular mechanism. In this study, we propose that neuronal calcium sensor 1 (NCS1), can be an effective molecular target for Li+ action. Here we show that the EF-hands in ApoNCS1 have submillimolar affinity for Li+ with Kd = 223 ± 19 μM. Li+ binding to ApoNCS1 quenches Trp emission intensity, suggesting distinct Trp sidechains environment in Li+NCS1 compared to ApoNCS1 and Ca2+NCS1. Li+ association also stabilizes the protein α-helical structure, in a similar way to Ca2+. Li+ association does not promote NCS1 dimerization. Association of Li+ increases NCS1 affinity for the D2R receptor binding peptide, in a similar way to Ca2+, however, the affinity of NCS1 for chlorpromazine is reduced with respect to Ca2+NCS1, possibly due to a decrease in solvent exposed hydrophobic area on the NCS1 surface in the presence of Li+. MD simulation data suggests that Li+ ions are coordinated by four oxygens from Asp and Glu sidechains and one carbonyl oxygen, in a similar way as reported previously for Li+ binding to DREAM. Overall, the data shows that Li+ binds to EF-hands of NCS1 and Li+NCS1 interactions may be involved in the potential neuroprotective role of Li+ against psychotic disorders.

Solid state NMR spectral editing of histidine, arginine and lysine using Hadamard encoding.

The NMR signals from protein sidechains are rich in information about intra- and inter-molecular interactions, but their detection can be complicated due to spectral overlap as well as conformational and hydrogen exchange. In this work, we demonstrate a protocol for multi-dimensional solid-state NMR spectral editing of signals from basic sidechains based on Hadamard matrix encoding. The Hadamard method acquires multi-dimensional experiments in such a way that both the backbone and under-sampled sidechain signals can be decoded for unambiguous editing in the 15 N spectral frequency dimension. All multi-dimensional 15 N-edited solid-state NMR experiments can be acquired using this strategy, thereby accelerating the acquisition of spectra spanning broad frequency bandwidth. Application of these methods to the ferritin nanocage, reveals signals from N atoms from His, Arg, Lys and Trp sidechains, as well as their tightly bound, ordered water molecules. The Hadamard approach adds to the arsenal of spectroscopic approaches for protein NMR signal detection.

Relationships between conformational and vibrational features of tryptophan characteristic Raman markers

Tryptophan (Trp) residue provides characteristic vibrational markers to the middle wavenumber spectral region of the Raman spectra recorded from peptides and proteins. In this report, we were particularly interested in eight Trp Raman markers, referred to as Wi (i = 1,…,8). All responsible for pronounced Raman lines, these markers originate from indole moiety, a bicyclic conjugated segment involved in the Trp structure. Numerous investigations have previously attempted to relate the variations observed in the spectral features of these markers to the environmental changes of Trp residues. To emphasize the most important points we can mention (i) the variations in the Raman profile of W4 (∼1360 cm−1) and W5 (∼1340 cm−1), frequently observed as a doublet with variable intensity ratio. These two markers were thought to result from a Fermi-resonance effect between certain planar and nonplanar modes; (ii) the changes observed in the wavenumbers and relative intensities of W4, W7 (∼880 cm−1) and W8 (∼760 cm−1) were supposed to be related to the accessibility of Trp to surrounding water molecules; and (iii) the wavenumber fluctuations of W3 (∼1550 cm−1), taken as a Trp side chain orientational marker. However, some ambiguities still exist regarding the interpretation of these markers, needing further clarification. Herein, upon a joint experimental and theoretical analysis based on a multiconformational approach, attention was paid to the relationships between structural and vibrational features of three indole-containing compounds with increasing structural complexity, i.e., skatole (3–methylindole), tryptophan, and tripeptide Gly–Trp–Gly. This study clearly shows that the existing assignments given to certain Trp Raman markers should be reconsidered, especially those based on the Fermi-resonance origin of W4-W5 (∼1360–1340 cm−1) doublet, as well as the purely environmental dependence of W7 and W8 markers.

Hordeum vulgareCYP76M57 catalyzes C2 shortening of tryptophan side chain by C-N bond rearrangement in gramine biosynthesis.

The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cβ. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.

Influence of amino acid and N-terminal protection residue structures on peptide p-nitroanilide adsorption on polystyrene-based support.

Polystyrene-based support Bio-Beads® SM-2 was employed for desalting peptide-p-nitroanilides from Oxone®. Neither tosyl, 9-fluorenyl(methoxycarbonyl), p-nitroanilide groups nor indolyl or p-hydroxyphenyl side-chains of Trp and Tyr ensured an efficient adsorption of peptide-p-nitroanilides onto Bio-Beads® SM-2. Only unsubstituted phenyl-containing protection groups (carbobenzoxy or benzoyl) and Phe residues provided the adsorption of peptides on Bio-Beads® SM-2 and their efficient desalting. This support is well suitable for multiple parallel phenyl group-containing peptide derivative separations and high-throughput screenings.

C—H...O bonds involving Trp sidechain in protein structures

C—H...O bonds involving Trp sidechain in protein structures

Teraryl Braces in Macrocycles: Synthesis and Conformational Landscape Remodeling of Peptides.

The three-dimensional structure of medium-sized cyclic peptides accounts for their biological activity and other important physiochemical properties. Despite significant advances in the past few decades, chemists' ability to fine-tune the structure, in particular, the backbone conformation, of short peptides made of canonical amino acids is still quite limited. Nature has shown that cross-linking the aromatic side chains of linear peptide precursors via enzyme catalysis can generate cyclophane-braced products with unusual structures and diverse activities. However, the biosynthetic path to these natural products is challenging to replicate in the synthetic laboratory using practical chemical modifications of peptides. Herein, we report a broadly applicable strategy to remodel the structure of homodetic peptides by cross-linking the aromatic side chains of Trp, His, and Tyr residues with various aryl linkers. The aryl linkers can be easily installed via copper-catalyzed double heteroatom-arylation reactions of peptides with aryl diiodides. These aromatic side chains and aryl linkers can be combined to form a large variety of assemblies of heteroatom-linked multi-aryl units. The assemblies can serve as tension-bearable multijoint braces to modulate the backbone conformation of peptides as an entry to previously inaccessible conformational space.

Origin of a Double-Band Feature in the Ethylenic C═C Stretching Modes of the Retinal Chromophore in Heliorhodopsins.

Photoreceptor proteins play a critical role in light utilization for energy conversion and environmental sensing. Rhodopsin is a prototypical photoreceptor protein containing a retinal group that functions as a light-receptive site. It is essential to characterize the structure of the retinal chromophore because the chromophore structure, along with retinal-protein interactions, regulates which wavelengths of light are absorbed. Resonance Raman spectroscopy is a powerful tool to characterize chromophore structures in proteins. The resonance Raman spectra of heliorhodopsins, a recently discovered rhodopsin family, were previously reported to exhibit two intense ethylenic C═C stretching bands never observed for type-1 rhodopsins. Here, we show that the double-band feature in the ethylenic C═C stretching modes is not due to structural inhomogeneity but rather to the retinal polyene chain's linear structure. It contrasts with bent all-trans chromophore in type-1 rhodopsins. The linear structure of the chromophore results from weak atomic contacts between the 13-methyl group and a nearby Trp side chain, which can slow thermal reisomerization in the photocycle. It is possible that the deceleration of reisomerization increases the lifetime of the signaling intermediate for photosensory function.

Bioinformatic Atlas of Radical SAM Enzyme-Modified RiPP Natural Products Reveals an Isoleucine-Tryptophan Crosslink.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing family of natural products with diverse activities and structures. RiPP classes are defined by the tailoring enzyme, which can introduce a narrow range of modifications or a diverse set of alterations. In the latter category, RiPPs synthesized by radical S-adenosylmethionine (SAM) enzymes, known as RaS-RiPPs, have emerged as especially divergent. A map of all RaS-RiPP gene clusters does not yet exist. Moreover, precursor peptides remain difficult to predict using computational methods. Herein, we have addressed these challenges and report a bioinformatic atlas of RaS-RiPP gene clusters in available microbial genome sequences. Using co-occurrence of RaS enzymes and transporters from varied families as a bioinformatic hook in conjunction with an in-house code to identify precursor peptides, we generated a map of ∼15,500 RaS-RiPP gene clusters, which reveal a remarkable diversity of syntenies pointing to a tremendous range of enzymatic and natural product chemistries that remain to be explored. To assess its utility, we examined one family of gene clusters encoding a YcaO enzyme and a RaS enzyme. We find the former is noncanonical, contains an iron-sulfur cluster, and installs a novel modification, a backbone amidine into the precursor peptide. The RaS enzyme was also found to install a new modification, a C-C crosslink between the unactivated terminal δ-methyl group of Ile and a Trp side chain. The co-occurrence search can be applied to other families of RiPPs, as we demonstrate with the emerging DUF692 di-iron enzyme superfamily.

Tandem Electrochemical Oxidative Azidation/Heterocyclization of Tryptophan‐Containing Peptides under Buffer Conditions

AbstractAs the aromatic tryptophan (Trp) side chain plays a pivotal role in influencing the structure and function of peptides and proteins, it has become an attractive target for the late‐stage modification of these important biomolecules. Herein, we report an electrochemical approach for late‐stage functionalization of peptides containing a Trp side chain through manganese‐catalyzed tandem radical azidation/heterocyclization. This electrochemical oxidative strategy provides access to azide‐substituted tetrazolo[1,5‐a]indole‐containing peptides with broad functional group tolerance, high site selectivity, and good yields of products (up to 87 %) under mild buffer conditions. Moreover, the modified Trp‐containing peptides bearing an azide functionality are promising building blocks, paving the way for the construction of various derivatives, such as “click” chemistry products.

Tandem Electrochemical Oxidative Azidation/Heterocyclization of Tryptophan-Containing Peptides under Buffer Conditions.

As the aromatic tryptophan (Trp) side chain plays a pivotal role in influencing the structure and function of peptides and proteins, it has become an attractive target for the late-stage modification of these important biomolecules. Herein, we report an electrochemical approach for late-stage functionalization of peptides containing a Trp side chain through manganese-catalyzed tandem radical azidation/heterocyclization. This electrochemical oxidative strategy provides access to azide-substituted tetrazolo[1,5-a]indole-containing peptides with broad functional group tolerance, high site selectivity, and good yields of products (up to 87 %) under mild buffer conditions. Moreover, the modified Trp-containing peptides bearing an azide functionality are promising building blocks, paving the way for the construction of various derivatives, such as "click" chemistry products.

Cyclic N-locked indolicidin analogues with antimicrobial activity: Effect of ring size and fatty acid acylation

Novel antimicrobial drugs are in high demand due to the increasing emergence of multi-drug resistant bacteria. In recent years, much attention has been given to natural occurring antimicrobial peptides (AMPs) such as indolicidin (ILPWKWPWWPWRR-NH2). In this study, 19 cyclic N-locked indolicidin analogues (3–21) were synthesized successfully by standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) combined with a convenient on-resin N-terminus to lysine side-chain cyclization via intramolecular halide substitution using bromoacetic acid. The effect of the ring size on antimicrobial activity and cytotoxicity was initially investigated and subsequently, the effect of fatty acid acylation was explored. We observed that a peptide macrocycle consisting of seven residues was optimal for Gram-positive antibacterial activity. Circular dichroism (CD) spectra of peptide macrocycles (3–13) showed that certain positions of ring-closure induced a conformation seen under sodium dodecyl sulfate (SDS) conditions where Trp side-chains can display exciton coupling. The far-UV CD was consistent with a backbone turn, a conformation with no evidence of helical structure. SAXS data on peptides mixed with lipid vesicles indicate a similar membrane interaction, when comparing the cyclic and linear indolicidin. The most potent analogue identified was 7, which showed antibacterial activity against Gram-positive bacteria (MIC against Staphylococcus aureus ATCC29213 ​= ​12.5 ​μg/mL and Staphylococcus epidermidis HJ56 ​= ​6.3 ​μg/mL). In addition, viable HaCaT cells after 200 ​μg/mL treatment with 7 was 84.3%. The results show that N-locked cyclization of linear antimicrobial peptides provides a method to reduce cytoxicity; although it may affect the antimicrobial activity.

Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix–coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

EXECUTER2 modulates the EXECUTER1 signalosome through its singlet oxygen-dependent oxidation

Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling. The Arabidopsis thaliana EXECUTER1 (EX1) protein, a chloroplast-localized singlet oxygen (1O2) sensor, undergoes tryptophan (Trp) 643 oxidation by 1O2, a chloroplast-derived and light-dependent reactive oxygen species. The indole side chain of Trp is vulnerable to 1O2, leading to the generation of oxidized Trp variants and priming EX1 for degradation by a membrane-bound FtsH protease. The perception of 1O2 via Trp643 oxidation and subsequent EX1 proteolysis facilitate chloroplast-to-nucleus retrograde signaling. In this study, we discovered that the EX1-like protein EX2 also undergoes 1O2-dependent Trp530 oxidation and FtsH-dependent turnover, which attenuates 1O2 signaling by decelerating EX1-Trp643 oxidation and subsequent EX1 degradation. Consistent with this finding, the loss of EX2 function reinforces EX1-dependent retrograde signaling by accelerating EX1-Trp643 oxidation and subsequent EX1 proteolysis, whereas overexpression of EX2 produces molecular phenotypes opposite to those observed in the loss–of- function mutants of EX2. Intriguingly, phylogenetic analysis suggests that EX2 may have emerged evolutionarily to attenuate the sensitivity of EX1 toward 1O2. Collectively, these results suggest that EX2 functions as a negative regulator of the EX1 signalosome through its own 1O2-dependent oxidation, providing a new mechanistic insight into the regulation of EX1-mediated 1O2 signaling.

Synthesis and Inhibition Activity Study of Triazinyl-Substituted Amino(alkyl)-benzenesulfonamide Conjugates with Polar and Hydrophobic Amino Acids as Inhibitors of Human Carbonic Anhydrases I, II, IV, IX, and XII.

Primary sulfonamide derivatives with various heterocycles represent the most widespread group of potential human carbonic anhydrase (hCA) inhibitors with high affinity and selectivity towards specific isozymes from the hCA family. In this work, new 4-aminomethyl- and aminoethyl-benzenesulfonamide derivatives with 1,3,5-triazine disubstituted with a pair of identical amino acids, possessing a polar (Ser, Thr, Asn, Gln) and non-polar (Ala, Tyr, Trp) side chain, have been synthesized. The optimized synthetic, purification, and isolation procedures provided several pronounced benefits such as a short reaction time (in sodium bicarbonate aqueous medium), satisfactory yields for the majority of new products (20.6–91.8%, average 60.4%), an effective, well defined semi-preparative RP-C18 liquid chromatography (LC) isolation of desired products with a high purity (>97%), as well as preservation of green chemistry principles. These newly synthesized conjugates, plus their 4-aminobenzenesulfonamide analogues prepared previously, have been investigated in in vitro inhibition studies towards hCA I, II, IV and tumor-associated isozymes IX and XII. The experimental results revealed the strongest inhibition of hCA XII with low nanomolar inhibitory constants (Kis) for the derivatives with amino acids possessing non-polar side chains (7.5–9.6 nM). Various derivatives were also promising for some other isozymes.

Dimerization of Cadherin-11 involves multi-site coupled unfolding and strand swapping

Cadherin extracellular domain 1 (EC1) mediates homophilic dimerization in adherens junctions. Conserved Trp2 and Trp4 residues in type II cadherins anchor the EC1 A strand intermolecularly in strand-swapped dimers. Herein, NMR spectroscopy is used to elucidate the roles of Trp2 and Trp4 in Cadherin-11 dimerization. The monomeric state, with the A strand and Trp side chains packed intramolecularly, is in equilibrium with sparsely populated partially and fully A-strand-exposed states, in which Trp2 (and Trp4, respectively) side-chain packing is disrupted. Exchange kinetics between the major state and the partially (fully) A-strand-exposed state is slow-intermediate (intermediate-fast). A separate very fast process exchanges ordered and random-coil BC-loop conformations with populations dependent on A-strand exposure and dimerization status. In addition, very slow processes connect the folded A-strand-exposed conformation to partially unfolded states, which may represent additional domain-swapping intermediates. The dimerization mechanism of type II cadherins is revealed as coupled folding and strand swapping.

Photo-induced fragmentation of tyrosine side chains in IgG4-Fc: Effect of protein sequence, conformation and glycan structure

This account summarizes recent work on photo-induced side chain cleavage reactions of Tyr in IgG4-Fc. These processes are initiated via photo-ionization of Trp to a Trp radical cation, followed by one-electron oxidation of Tyr to a Tyr radical cation, where fragmentation of the Cα-Cβ bond leads to an intermediary glycyl radical. The latter converts into Gly via hydrogen atom transfer, or into various backbone cleavage products subsequent to the addition of oxygen. Site-directed mutation of Trp381 to Phe381 significantly reduces the yields of Tyr side chain cleavage products, underlining a critical role of Trp for these reactions. Likewise, conformational changes of IgG4-Fc induced by increasing contents of 1-propanol in the solution diminishes the yields of Tyr side chain cleavage products. Variations of the glycan structure at Asn297 had an effect on Tyr side chain cleavage, where larger glycan structures led to lower yields. This result is in interesting contrast to Trp side chain cleavage in IgG1-Fc, where larger glycan structures led to higher yields. The opposite trends for Trp and Tyr side chain cleavage as a function of glycan structure are consistent with a connection between the precursors for both reactions, radical cations of Trp and Tyr, related by electron transfer. The potential significance of these reactions for protein integrity in formulations of pharmaceutical proteins will be discussed.

Flexibility and Cell Permeability of Cyclic Ras-Inhibitor Peptides Revealed by the Coupled Nosé-Hoover Equation.

Quantifying the cell permeability of cyclic peptides is crucial for their rational drug design. However, the reasons remain unclear why a minor chemical modification, such as the difference between Ras inhibitors cyclorasin 9A5 and 9A54, can substantially change a peptide's permeability. To address this question, we performed enhanced sampling simulations of these two 11-mer peptides using the coupled Nosé-Hoover equation (cNH) we recently developed. The present cNH simulations realized temperature fluctuations over a wide range (240-600 K) in a dynamic manner, allowing structural samplings that were well validated by nuclear Overhauser effect measurements. The derived structural ensembles were comprehensively analyzed by all-atom structural clustering, mapping the derived clusters onto principal components (PCs) that characterize the cyclic structure, and calculating cluster-dependent geometric and chemical properties. The planar-open conformation was dominant in aqueous solvent, owing to inclusion of the Trp side chain in the main-chain ring, while the compact-closed conformation, which favors cell permeation due to its compactness and high polarity, was also accessible. Conformation-dependent cell permeability was observed in one of the derived PCs, demonstrating that decreased cell permeability in 9A54 is due to the high free energy barrier separating the two conformations. The origin of the change in free energy surface was determined to be loss of flexibility in the modified residues 2-3, resulting from the increased bulkiness of their side chains. The derived molecular mechanism of cell permeability highlights the significance of complete structural dynamics surveys for accelerating drug development with cyclic peptides.

Glycine rich segments adopt polyproline II helices: Implications for biomolecular condensate formation

Many intrinsically disordered proteins contain Gly-rich regions which are generally assumed to be disordered. Such regions often form biomolecular condensates which play essential roles in organizing cellular processes. However, the bases of their formation and stability are still not completely understood. Based on NMR studies of the Gly-rich H. harveyi “snow flea” antifreeze protein, we recently proposed that Gly-rich sequences, such as the third “RGG” region of Fused in Sarcoma (FUS) protein, may adopt polyproline II helices whose association might stabilize condensates. Here, this hypothesis is tested with a polypeptide corresponding to the third RGG region of FUS. NMR spectroscopy and molecular dynamics simulations suggest that significant populations of polyproline II helix are present. These findings are corroborated in a model peptide Ac-RGGYGGRGGWGGRGGY-NH2, where a peak characteristic of polyproline II helix is observed using CD spectroscopy. Its intensity suggests a polyproline II population of 40%. This result is supported by data from FTIR and NMR spectroscopies. In the latter, NOE correlations are observed between the Tyr and Arg, and Arg and Trp side chain hydrogens, confirming that side chains spaced three residues apart are close in space. Taken together, the data are consistent with a polyproline II helix, which is bent to optimize interactions between guanidinium and aromatic moieties, in equilibrium with a statistical coil ensemble. These results lend credence to the hypothesis that Gly-rich segments of disordered proteins may form polyproline II helices which help stabilize biomolecular condensates.