Genome wide identification of novel DNA methylation driven prognostic markers in colorectal cancer.

Colorectal cancer (CRC) stands as a major contributor to cancer-related fatalities within China. There is an urgent need to identify accurate biomarkers for recurrence predicting in CRC. Reduced representation bisulfite sequencing was used to perform a comparative analysis of methylation profiles in tissue samples from 30 recurrence to 30 non-recurrence patients with CRC. Least absolute shrinkage and selection operator method was performed to select the differential methylation regions (DMRs) and built a DNA methylation classifier for predicting recurrence. Based on the identified top DMRs, a methylation classifier was built and consisted of eight hypermethylated DMRs in CRC. The DNA methylation classifier showed high accuracy for predicting recurrence with an area under the receiver operator characteristic curve of 0.825 (95% CI 0.680-0.970). The Kaplan-Meier survival analysis demonstrated that CRC patients with high methylation risk score, evaluated by the DNA methylation classifier, had poorer survival than low risk score (Hazard Ratio 4.349; 95% CI 1.783-10.61, P = 0.002). And only CRC patients with low methylation risk score could acquire benefit from adjuvant therapy. The DNA methylation classifier has been proved as crucial biomarkers for predicting recurrence and exhibited promising prognostic value after curative surgery in patients with CRC.

Mitochondrial dynamics dysfunction: Unraveling the hidden link to depression.

Depression is a common mental disorder and its pathogenesis is not fully understood. However, more and more evidence shows that mitochondrial dynamics dysfunction may play an important role in the occurrence and development of depression. Mitochondria are the centre of energy production in cells, and are also involved in important processes such as apoptosis and oxidative stress. Studies have found that there are abnormalities in mitochondrial function in patients with depression, including mitochondrial morphological changes, mitochondrial dynamics disorders, mitochondrial DNA damage, and impaired mitochondrial respiratory chain function. These abnormalities may cause excessive free radicals and oxidative stress in mitochondria, which further damage cells and affect the balance of neurotransmitters, causing or aggravating depressive symptoms. Studies have shown that mitochondrial dynamics dysfunction may participate in the occurrence and development of depression by affecting neuroplasticity, inflammation and neurotransmitters. This article reviews the effects of mitochondrial dynamics dysfunction on the pathogenesis of depression and its potential molecular pathway. The restorers for the treatment of depression by regulating the function of mitochondrial dynamics were summarized and the possibility of using mitochondrial dynamics as a biomarker of depression was discussed.

NONO promotes gallbladder cancer cell proliferation by enhancing oncogenic RNA splicing of DLG1 through interaction with IGF2BP3/RBM14.

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment.

Plant species identity and mycorrhizal type explain the root-associated fungal pathogen community assembly of seedlings based on functional traits in a subtropical forest.

Introduction: As a crucial factor in determining ecosystem functioning, interaction between plants and soil-borne fungal pathogens deserves considerable attention. However, little attention has been paid into the determinants of root-associated fungal pathogens in subtropical seedlings, especially the influence of different mycorrhizal plants. Methods: Using high-throughput sequencing techniques, we analyzed the root-associated fungal pathogen community for 19 subtropical forest species, including 10 ectomycorrhizal plants and 9 arbuscular mycorrhizal plants. We identified the roles of different factors in determining the root-associated fungal pathogen community. Further, we identified the community assembly process at species and mycorrhizal level and managed to reveal the drivers underlying the community assembly. Results: We found that plant species identity, plant habitat, and plant mycorrhizal type accounted for the variations in fungal pathogen community composition, with species identity and mycorrhizal type showing dominant effects. The relative importance of different community assembly processes, mainly, homogeneous selection and drift, varied with plant species identity. Interestingly, functional traits associated with acquisitive resource-use strategy tended to promote the relative importance of homogeneous selection, while traits associated with conservative resource-use strategy showed converse effect. Drift showed the opposite relationships with functional traits compared with homogeneous selection. Notably, the relative importance of different community assembly processes was not structured by plant phylogeny. Drift was stronger in the pathogen community for ectomycorrhizal plants with more conservative traits, suggesting the predominant role of stochastic gain and loss in the community assembly. Discussion: Our work demonstrates the determinants of root-associated fungal pathogens, addressing the important roles of plant species identity and plant mycorrhizal type. Furthermore, we explored the community assembly mechanisms of root-associated pathogens and stressed the determinant roles of functional traits, especially leaf phosphorus content (LP), root nitrogen content (RN) and root tissue density (RTD), at species and mycorrhizal type levels, offering new perspectives on the microbial dynamics underlying ecosystem functioning.

Host Recognition and Specific Infection of Endomelanconiopsis endophytica during Early Infection.

Coevolution between the pathogen and host plant drives pathogenic effector diversity. However, the molecular mechanism behind host-specific pathogenesis remains to be explored. Here, we present a 43 Mb whole-genome sequence of Endomelanconiopsis endophytica strain LS29, a host-specific pathogen of the common subtropical tree Castanopsis fissa. We described its genome annotations and identified its effector candidates. By performing temporal transcriptome sequencing of E. endophytica on C. fissa during early infection, we found that E. endophytica repressed other microbes in order to attack the tissue of the host by producing antibiotics earlier than 24 h post-inoculation (hpi). Simultaneously, a variety of effectors were secreted to recognize the host plant, but most of them showed a significantly opposing expression regulation trend after 24 hpi, indicating that 24 hpi represents a key time point between host recognition and specific infection. Furthermore, a comparison of isoenzymes showed that only a few effectors were identified as specific effectors, which were involved in hydrolyzing the compounds of the plant cell wall and releasing fatty acids during the early infection of C. fissa. Our results determined host recognition timing and identified a specific catalog of effectors, which are crucial for revealing the molecular mechanism of host-specific pathogenesis.

Ponicidin inhibited gallbladder cancer proliferation and metastasis by decreasing MAGEB2 expression through FOXO4.

BACKGROUND: Gallbladder cancer (GBC) is the most aggressively malignant tumor in the bile duct system. The prognosis for patients with GBC is extremely poor. Ponicidin is a diterpenoid compound extracted and purified from the traditional Chinese herb Rabdosia rubescens, and showed promising anti-cancer effects in a variety of tumors. However, Ponicidin has not been investigated in GBC. METHODS: CCK-8, colony formation assay and EdU-488 DNA synthesis assay were performed to investigate the effect of Ponicidin on GBC cells proliferation. Cell invasion and migration assays and wound-healing assay were used to explore the effect of Ponicidin on invasion and migration ability of GBC cells. mRNA-seq was adopted to explore the underlying mechanisms. Western blot and immunohistochemical staining were conducted to detect the protein level. CHIP assay and dual-luciferase assay were used to validate binding motif. Nude mouse model of GBC was used to assess the anti-tumor effect and safety of Ponicidin. RESULTS: Ponicidin inhibited the proliferation and cell invasion and migration of GBC cells in vitro. Moreover, Ponicidin exerted anti-tumor effects by down-regulating the expression of MAGEB2. Mechanically, Ponicidin upregulated the FOXO4 expression and promoted it to accumulate in nucleus to inhibit the transcript of MAGEB2. Furthermore, Ponicidin suppressed tumor growth in the nude mouse model of GBC with excellent safety. CONCLUSION: Ponicidin may be a promising agent for the treatment of GBC effectively and safely.

Exosomes may be the carrier of acupuncture treatment for major depressive disorder.

The incidence of major depressive disorder (MDD) is increasing all over the world. There is a great need for complementary or alternative therapies with high safety, few side effects, and precise efficacy to care for MDD. In China, acupuncture has significant laboratory data and clinical trials to demonstrate its antidepressant efficacy. However, there is no clear answer as to how it works. Exosomes are membranous vesicles that rely on cellular multivesicular bodies (MVBs) fused to the cell membrane for release into the extracellular matrix. Almost all cell types are capable of producing and releasing exosomes. As a result, exosomes contain complex RNAs and proteins from their relatives (Cells that secretes exosomes). They can cross biological barriers and participate in biological activities, such as cell migration, angiogenesis, and immune regulation. These properties have made them a popular research topic. Some experts have suggested that exosomes may serve as delivery vehicles for acupuncture to work. This presents both an opportunity and a new challenge for improving the protocols of acupuncture as a treatment for MDD. To better define the relationship between MDD, exosomes, and acupuncture, we reviewed the literature from the last few years. Inclusion criteria included randomized controlled trials and basic trials evaluating acupuncture in the treatment or prevention of MDD, the role of exosomes in the development and progression of MDD, and the role of exosomes in acupuncture. We believe that acupuncture may affect the distribution of exosomes in vivo, and exosomes may be a new carrier for acupuncture treatment of MDD in the future.

Soil fungal networks moderate density-dependent survival and growth of seedlings.

Pathogenic and mutualistic fungi have contrasting effects on seedling establishment, but it remains unclear whether density-dependent survival and growth are regulated by access to different types of mycorrhizal fungal networks supported by neighbouring adult trees. Here, we conducted an extensive field survey to test how mycorrhizal and pathogenic fungal colonization of arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) seedlings in a subtropical forest respond to density of neighbouring adult trees. In addition, we undertook a hyphal exclusion experiment to explicitly test the role of soil fungal networks in driving density-dependent effects on seedling growth and survival. Conspecific adult density was a strong predictor for the relative abundance of putative pathogens, which was greater in roots of AM than of ECM seedlings, while mycorrhizal fungal abundance and colonization were not consistently affected by conspecific adult density. Both ECM and AM fungal networks counteracted conspecific density-dependent mortality, but ECM fungi were more effective at weakening the negative effects of high seedling density than AM fungi. Our findings reveal a critical role of common fungal networks in mitigating negative density-dependent effects of pathogenic fungi on seedling establishment, which provides mechanistic insights into how soil fungal diversity shapes plant community structure in subtropical forests.

Mechanistic Studies of the Kinase Domains of Class IV Lanthipeptide Synthetases.

Lanthipeptides, which belong to the superfamily of ribosomally synthesized and posttranslationally modified peptides (RiPPs), are associated with various interesting biological activities. Lanthipeptides can be subdivided into four classes that are defined by the characteristics of the corresponding posttranslational modification enzymes. Class IV lanthipeptide synthetases consist of an N-terminal lyase, a central kinase, and a C-terminal cyclase domain. Here, we present the first in-depth characterization of such a kinase domain from the globisporin maturation enzyme SgbL that originates from Streptomyces globisporus sp. NRRL B-2293. Catalytic residues were identified by alignments with homologues and structural modeling. Their roles were confirmed by employing proteins with Ala substitutions in in vitro modification and fluorescence polarization binding assays. Furthermore, the protein region that is binding the leader peptide was identified by hydrogen-deuterium exchange-mass spectrometry experiments. By fusion of this protein region to the maltose binding protein, a protein was generated that can specifically bind the SgbA leader peptide, albeit with reduced binding affinity compared to that of full length SgbL. Combined, the results of this study provide a firmer grasp of how lanthipeptide biosynthesis is accomplished by class IV synthetases and suggest by homology analysis that biosynthetic mechanisms are similar in class III lanthipeptide processing enzymes.

Inhibition of Marburg Virus RNA Synthesis by a Synthetic Anti-VP35 Antibody.

Marburg virus causes sporadic outbreaks of severe hemorrhagic fever with high case fatality rates. Approved, effective, and safe therapeutic or prophylactic countermeasures are lacking. To address this, we used phage display to engineer a synthetic antibody, sFab H3, which binds the Marburg virus VP35 protein (mVP35). mVP35 is a critical cofactor of the viral replication complex and a viral immune antagonist. sFab H3 displayed high specificity for mVP35 and not for the closely related Ebola virus VP35. sFab H3 inhibited viral-RNA synthesis in a minigenome assay, suggesting its potential use as an antiviral. We characterized sFab H3 by a combination of biophysical and biochemical methods, and a crystal structure of the complex solved to 1.7 Å resolution defined the molecular interface between the sFab H3 and mVP35 interferon inhibitory domain. Our study identifies mVP35 as a therapeutic target using an approach that provides a framework for generating engineered Fabs targeting other viral proteins.

Recognition of Human IgG1 by Fcγ Receptors: Structural Insights from Hydrogen-Deuterium Exchange and Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an effector function of immunoglobulins (IgGs) involved in the killing of target cells by a cytotoxic effector cell. Recognition of IgG by Fc receptors expressed on natural killer cells, mostly FcγIII receptors (FcγRIII), underpins the ADCC mechanism, thus motivating investigations of these interactions. In this paper, we describe the combination of hydrogen-deuterium exchange and fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry to study the interactions of the human IgG1/FcγRIII complex. Using these orthogonal approaches, we identified critical peptide regions and residues involved in the recognition of IgG1 by FcγRIII. The footprinting results are consistent with the previously published crystal structure of the IgG1 Fc/FcγRIII complex. Additionally, our FPOP results reveal the conformational changes in the Fab domain upon binding of the Fc domain to FcγRIII. These data demonstrate the value of footprinting as part of a comprehensive toolbox for identifying the changes in the higher-order structure of therapeutic antibodies in solution.

Fast Photochemical Oxidation of Proteins Coupled with Mass Spectrometry.

BACKGROUND: Determination of the composition and some structural features of macromolecules can be achieved by using structural proteomics approaches coupled with mass spectrometry (MS). One approach is hydroxyl radical protein footprinting whereby amino-acid side chains are modified with reactive reagents to modify irreversibly a protein side chain. The outcomes, when deciphered with mass-spectrometry-based proteomics, can increase our knowledge of structure, assembly, and conformational dynamics of macromolecules in solution. Generating the hydroxyl radicals by laser irradiation, Hambly and Gross developed the approach of Fast Photochemical Oxidation of Proteins (FPOP), which labels proteins on the sub millisecond time scale and provides, with MS analysis, deeper understanding of protein structure and protein-ligand and protein- protein interactions. This review highlights the fundamentals of FPOP and provides descriptions of hydroxyl-radical and other radical and carbene generation, of the hydroxyl labeling of proteins, and of determination of protein modification sites. We also summarize some recent applications of FPOP coupled with MS in protein footprinting. CONCLUSION: We survey results that show the capability of FPOP for qualitatively measuring protein solvent accessibility on the residue level. To make these approaches more valuable, we describe recent method developments that increase FPOP's quantitative capacity and increase the spatial protein sequence coverage. To improve FPOP further, several new labeling reagents including carbenes and other radicals have been developed. These growing improvements will allow oxidative- footprinting methods coupled with MS to play an increasingly significant role in determining the structure and dynamics of macromolecules and their assemblies.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly.

Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

Mass Spectrometry-Based Fast Photochemical Oxidation of Proteins (FPOP) for Higher Order Structure Characterization.

Assessment of protein structure and interaction is crucial for understanding protein structure/function relationships. Compared to high-resolution structural tools, including X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-EM, and traditional low-resolution methods, such as circular dichroism, UV-vis, and florescence spectroscopy, mass spectrometry (MS)-based protein footprinting affords medium-to-high resolution (i.e., regional and residue-specific insights) by taking advantage of proteomics methods focused on the primary structure. The methodology relies on "painting" the reactive and solvent-exposed amino acid residues with chemical tags and using the pattern of modifications as footprints from analysis by bottom-up MS-based proteomics to deduce protein higher order structures. The outcome can refer to proteins in solution or even in cells and is complementary to those of X-ray crystallography and NMR. It is particularly useful in mapping protein-ligand interfaces and conformational changes resulting from ligand binding, mutation, and aggregation. Fast photochemical oxidation of proteins (FPOP), in its original conception, is a type of hydroxyl-radical-based protein footprinting that utilizes a pulsed KrF laser (248 nm) to trigger hydrolysis of hydrogen peroxide to produce solution hydroxyl radicals, which subsequently modify the protein in situ. The platform is expanding to adopt other reactive species including carbenes. The reactivity of the probe depends on the intrinsic reactivity of the radical with the residue side chain and the solvent accessibility of the residue as a function of the tertiary/quaternary structures. By introducing an appropriate scavenger to compete with hydroxyl radical self-quenching, the lifetime of the primary radicals is remarkably shortened to approximately microsecond. Thus, the sampling time scale of FPOP is much faster than hydrogen-deuterium exchange and other covalent labeling methods relying on nonradical reactions. The short footprinting time scale of FPOP offers two major advantages for protein structure elucidation: (1) it allows the protein to be interrogated in its native or near-native state with minimum structural perturbation; (2) it exhibits high sensitivity toward alterations in protein higher order structures because its sampling time is short with respect to protein conformational changes and dynamic motion. In addition, the covalent and irreversible oxidation by the hydroxyl radical provides more flexibility in the downstream proteomics workflow and MS analysis, permitting high spatial resolution with residue-specific information. Since its invention in 2005 by Hambly and Gross, FPOP has developed from proof-of-concept to a valuable biophysical tool for interrogating protein structure. In this Account, we summarize the principles and experimental design of FPOP that enable its fast labeling and describe the current and unique capabilities of the technique in protein higher order structure elucidation. Application examples include characterization of amyloid ß self-assembly, protein-ligand interactions with a special emphasis on epitope mapping for protein therapeutics (e.g., antibody, Fab, and adnectin), protein folding detailed to residue-specific folding kinetics, and protein flexibility/dynamics. Additionally, the utility of FPOP-based oxidative footprinting should grow with our continuing developments of novel reagents (e.g., sulfate radical anion, carbene diradical, and trifluoromethyl radical). These reactive reagents are compatible with the current FPOP platform and offer different reactivity and selectivity toward various types of amino acid residues, providing complementary insights into protein higher order structures for soluble proteins and ultimately for membrane-bound proteins.

A Molecular Mechanism for Nonphotochemical Quenching in Cyanobacteria.

The cyanobacterial orange carotenoid protein (OCP) protects photosynthetic cyanobacteria from photodamage by dissipating excess excitation energy collected by phycobilisomes (PBS) as heat. Dissociation of the PBS-OCP complex in vivo is facilitated by another protein known as the fluorescence recovery protein (FRP), which primarily exists as a dimeric complex. We used various mass spectrometry (MS)-based techniques to investigate the molecular mechanism of this FRP-mediated process. FRP in the dimeric state (dFRP) retains its high affinity for the C-terminal domain (CTD) of OCP in the red state (OCPr). Site-directed mutagenesis and native MS suggest the head region on FRP is a candidate to bind OCP. After attachment to the CTD, the conformational changes of dFRP allow it to bridge the two domains, facilitating the reversion of OCPr into the orange state (OCPo) accompanied by a structural rearrangement of dFRP. Interestingly, we found a mutual response between FRP and OCP; that is, FRP and OCPr destabilize each other, whereas FRP and OCPo stabilize each other. A detailed mechanism of FRP function is proposed on the basis of the experimental results.

Native Mass Spectrometry Analysis of Oligomerization States of Fluorescence Recovery Protein and Orange Carotenoid Protein: Two Proteins Involved in the Cyanobacterial Photoprotection Cycle.

The orange carotenoid protein (OCP) and fluorescence recovery protein (FRP) are present in many cyanobacteria and regulate an essential photoprotection cycle in an antagonistic manner as a function of light intensity. We characterized the oligomerization states of OCP and FRP by using native mass spectrometry, a technique that has the capability of studying native proteins under a wide range of protein concentrations and molecular masses. We found that dimeric FRP is the predominant state at protein concentrations ranging from 3 to 180 µM and that higher-order oligomers gradually form at protein concentrations above this range. The OCP, however, demonstrates significantly different oligomerization behavior. Monomeric OCP (mOCP) dominates at low protein concentrations, with an observable population of dimeric OCP (dOCP). The ratio of dOCP to mOCP, however, increases proportionally with protein concentration. Higher-order OCP oligomers form at protein concentrations beyond 10 µM. Additionally, native mass spectrometry coupled with ion mobility allowed us to measure protein collisional cross sections and interrogate the unfolding of different FRP and OCP oligomers. We found that monomeric FRP exhibits a one-stage unfolding process, which could be correlated with its C-terminal bent crystal structure. The structural domain compositions of FRP and OCP are compared and discussed.

Following a Folding Transition with Capillary Electrophoresis and Ion Mobility Spectrometry.

Ion mobility spectrometry (IMS) is increasingly used to describe solution-phase phenomena and has recently been used to establish the presence of multiple intermediates during the folding of a model polypeptide, polyproline. These observations, however, are made on gas-phase structures. Capillary electrophoresis (CE) is a complementary solution-phase technique, also based on the separation of charged species as a function of size and charge. Here, both ion mobility and capillary electrophoresis are used to follow the folding transition of a 13-mer polyproline peptide from the all-cis polyproline I (PPI) conformation to the all-trans polyproline II (PPII) conformation upon immersion in aqueous solvent. Synchronous folding processes are observed using both techniques. Eight conformers are observed using ion mobility. Although only five peaks are observed using capillary electrophoresis, these peaks can be modeled as sums of the observed IMS conformers; this is strong evidence that ion mobility is sampling solution-phase structures. CE measurements provide the first direct evidence that multiple folding intermediates are present in solution.

"Wet" Versus "Dry" Folding of Polyproline.

When the all-cis polyproline-I helix (PPI, favored in 1-propanol) of polyproline-13 is introduced into water, it folds into the all-trans polyproline-II (PPII) helix through at least six intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we show that the solvent-free intermediates refold into the all-cis PPI helix with high (>90%) efficiency. Moreover, in the absence of solvent, each intermediate appears to utilize the same small set of pathways observed for the solution-phase PPII → PPI transition upon immersion of PPIIaq in 1-propanol. That folding in solution (under conditions where water is displaced by propanol) and folding in vacuo (where energy required for folding is provided by collisional activation) occur along the same pathway is remarkable. Implicit in this statement is that 1-propanol mimics a "dry" environment, similar to the gas phase. We note that intermediates with structures that are similar to PPIIaq can form PPII under the most gentle activation conditions-indicating that some transitions observed in water (i.e., "wet" folding, are accessible (albeit inefficient) in vacuo. Lastly, these "dry" folding experiments show that PPI (all cis) is favored under "dry" conditions, which underscores the role of water as the major factor promoting preference for trans proline. Graphical Abstract ᅟ.

Ion Mobility-Mass Spectrometry Reveals the Energetics of Intermediates that Guide Polyproline Folding.

Proline favors trans-configured peptide bonds in native proteins. Although cis/trans configurations vary for non-native and unstructured states, solvent also influences these preferences. Water induces the all-cis right-handed polyproline-I (PPI) helix of polyproline to fold into the all-trans left-handed polyproline-II (PPII) helix. Our recent work has shown that this occurs via a sequential mechanism involving six resolved intermediates [Shi, L., Holliday, A.E., Shi, H., Zhu, F., Ewing, M.A., Russell, D.H., Clemmer, D.E.: Characterizing intermediates along the transition from PPI to PPII using ion mobility-mass spectrometry. J. Am. Chem. Soc. 136, 12702-12711 (2014)]. Here, we use ion mobility-mass spectrometry to make the first detailed thermodynamic measurements of the folding intermediates, which inform us about how and why this transition occurs. It appears that early intermediates are energetically favorable because of the hydration of the peptide backbone, whereas late intermediates are enthalpically unfavorable. However, folding continues, as the entropy of the system increases upon successive formation of each new structure. When PPII is immersed in 1-propanol, the PPII→PPI transition occurs, but this reaction occurs through a very different mechanism. Early on, the PPII population splits onto multiple pathways that eventually converge through a late intermediate that continues on to the folded PPI helix. Nearly every step is endothermic. Folding results from a stepwise increase in the disorder of the system, allowing a wide-scale search for a critical late intermediate. Overall, the data presented here allow us to establish the first experimentally determined energy surface for biopolymer folding as a function of solution environment.