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[Display omitted] • 13C from PBAT monomers was tracked under anaerobic and thermophilic conditions. • 1,4-butanediol and adipic acid were assimilated in the biomass. • 3 genera were able to directly metabolise at least 1 of the 3 PBAT monomer units. • A HQ-MAG was identified as A. thermophila and as an active degrader of butanediol. PBAT (poly butylene adipate-co-terephthalate) is a widely used biodegradable plastic, but the knowledge about its metabolization in anaerobic environments is very limited. In this study, the anaerobic digester sludge from a municipal wastewater treatment plant was used as inoculum to investigate the biodegradability of PBAT monomers in thermophilic conditions. The research employs a combination of 13C-labelled monomers and proteogenomics to track the labelled carbon and identify the microorganisms involved. A total of 122 labelled peptides of interest were identified for adipic acid (AA) and 1,4-butanedio (BD). Through the time-dependent isotopic enrichment and isotopic profile distributions, Bacteroides , Ichthyobacterium , and Methanosarcina were proven to be directly involved in the metabolization of at least one monomer. This study provides a first insight into the identity and genomic potential of microorganisms responsible for biodegradability of PBAT monomers during anaerobic digestion under thermophilic conditions. [ABSTRACT FROM AUTHOR]

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaMF142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaMF142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142LCaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. [ABSTRACT FROM AUTHOR]

Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. 13C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using ³H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions. [ABSTRACT FROM AUTHOR]

Abstract: Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an α-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8–C16 alkyl chains and the inclusion of anionic lipids both increase Nc''s ability to form α-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of α-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of α-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing “plugging” effect. [Copyright &y& Elsevier]

Abstract: Polymers imprinted with sucrose and corresponding non-imprinted polymers are prepared photo-chemically at 3°C and thermally at 65°C. The pre-polymerization complex formation in dimethyl sulfoxide between sucrose and methacrylic acid via hydrogen bonding was investigated through 1H NMR titration. The imprinting effect and the selectivity of the imprinted polymers in water are studied by batch rebinding studies with different mono and disaccharides and fitted to the Freundlich isotherm. Based on the calculated numbers of binding sites and average affinity, it is concluded that sucrose has been successfully imprinted at 3 and 65°C. The polymer imprinted at 3°C possesses the best recognition properties. The imprinted polymers are selective towards sucrose in water. [Copyright &y& Elsevier]

Abstract: Xylitol fatty acid esters of increasing chain length were synthesised from xylitol and free fatty acids in binary mixtures of tert-butanol (t-BuOH) and pyridine using Candida antarctica lipase B as biocatalyst. NMR and GC analyses revealed that monoesters of all acyl donors were primarily substituted at the C-1 position (93–97%, mol/mol) and to a minor extent at the C-2 position introducing asymmetry to the structure; while acylation at C-3 was not found. Diesters were esterified mainly at the C-1 and C-5 positions (87–100%, mol/mol) and to a minor extent 1,2-di-O-acyl- and 1,4-di-O-acyl xylitol esters were identified. Acylation of the secondary alcohol groups was interpreted as a result of acyl migration, because it occurred at a later stage of the process at a significantly lower rate than acylation of the primary alcohol groups and because it decreased with increasing chain length of the acyl donor. For acyl donors of chain lengths higher than four, the degree of esterification was controlled by the pyridine content of the reaction medium with the highest yield of monoesters obtained in 55% (v/v) pyridine. With butanoic acid the highest yield of monoester was obtained in pure t-BuOH. Under these conditions a degree of conversion of 26–41% of xylitol – and a yield of 22–29% monoester was obtained already after 4.5h of incubation at 45°C. The initial reaction rate for monoester synthesis was 5–19 times higher than for diester synthesis correlating inversely with the chain length of the acyl donor. At an acyl acceptor to donor ratio of 1:5 (mol/mol) the highest initial reaction rate of monoester synthesis was observed with hexanoic acid. [Copyright &y& Elsevier]

• Heterologous production of the lipopeptide W493 and fungal cytokinins in F. graminearum. • Yeast recombination enables reconstruction of large fungal gene clusters. • Intact 10 and 54 kb foreign gene clusters were transformed via ATMT or PMT. Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite's biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens -mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis– and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products. [ABSTRACT FROM AUTHOR]

Peptide-level quantification using mass spectrometry (MS) is no trivial task as the physicochemical properties affect both response and detectability. The specific amino acid (AA) sequence affects these properties, however the connection between sequence and intensity output remains poorly understood. In this work, we explore combinations of amino acid pairs (i.e., dimer motifs) to determine a potential relationship between the local amino acid environment and MS1 intensity. For this purpose, a deep learning (DL) model, consisting of an encoder-decoder with an attention mechanism, was built. The attention mechanism allowed to identify the most relevant motifs. Specific patterns were consistently observed where a bulky/aromatic and hydrophobic AA followed by a cationic AA as well as consecutive bulky/aromatic and hydrophobic AAs were found important for the prediction of the MS1 intensity. Correlating attention weights to mean MS1 intensities revealed that some important motifs, particularly containing Trp, His, and Cys, were linked with low responding peptides whereas motifs containing Lys and most bulky hydrophobic AAs were often associated with high responding peptides. Moreover, Asn-Gly was associated with low response. The model predicts MS1 response with a mean average percentage error of ∼11 % and a Pearson correlation coefficient of ∼0.64. While dimer representation of peptide sequences did not improve predictive capacity compared to single AA representation in earlier work, this work adds valuable insight for a better understanding of peptide response in MS analysis. SIGNIFICANCE: Mass spectrometry is not inherently quantitative, and the response of a compound relies not only on its concentration but also on the molecular composition. For mass spectrometry-based analysis of peptides, such as in bottom-up proteomics, this directly implies that the response cannot be used directly to quantify individual peptides. Moreover, the dependency of the response on the amino acid sequence of individual peptides remains poorly understood. Using a deep learning model based on a recurrent neural network with an attention mechanism, we here investigate how the presence of dimer motifs within a peptide affects the MS1 response through the analysis of intended equimolar peptide pools comprising almost 200,000 unique peptides in total. Not only do we identify certain dimer classes and specific dimers that substantially affect the MS1 response, but the model is also able to predict peptide intensity with low error rates within the independent test subset. The findings not only improve our understanding of the link between sequence and response for peptides but also highlight the potential of utilizing deep learning for developing methods allowing for absolute, label-free peptide quantification., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Poly(ethylene 2,5-furanoate) (PEF) is considered to be the next-generation green polyester and is hailed as a rising star among novel plastics. It is biobased, is nontoxic, and has comparable or improved properties compared to polyethylene terephthalate (PET). Biobased PEF offers lower life-cycle greenhouse gas emissions than PET. However, with its industrial production starting soon, relatively little is known about its actual recyclability. This work reports on the near complete depolymerization of PEF using two efficient PET hydrolases, FastPETase and leaf compost-cutinase (LCC), at loadings 4.5-17 times lower than previously reported. FastPETase and LCC exhibited maximum depolymerization of PEF, measured by weight loss and 2,5-furandicarboxylic acid (FDCA) production, using potassium phosphate-NaOH buffer at 50 and 65 °C, respectively. The 98% depolymerization of 13 g L -1 PEF film was achieved by three additions of the LCC in 72 h, while 78% weight loss was obtained using FastPETase in controlled conditions. Nonetheless, 92% weight loss was obtained with FastPETase when using only 6 g L -1 PEF. The main reaction products were identified as FDCA, ethylene glycol, and mono(2-hydroxyethyl)-furanoate. LCC performed better than FastPETase, in terms of both FDCA release and weight loss. The effect of crystallinity was evident on the enzymes' performance, as only 4% to 7% weight loss of crystalline PEF (32%) was recorded. Microscopy studies of the treated PEF films provided information on the surface erosion processes and revealed higher resistance of the crystalline phase, explaining the low level of depolymerization. The study presents important insights into the enzymatic hydrolysis of biobased PEF material and paves the path toward more viable applications within biopolymer waste recycling., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Mutations in the small, calcium-sensing, protein calmodulin cause cardiac arrhythmia and can ultimately prove lethal. Here, we report the impact of the G113R variant on the structure and dynamics of the calmodulin molecule, both in the presence and in the absence of calcium. We show that the mutation introduces minor changes into the structure of calmodulin and that it changes the thermostability and thus the degree of foldedness at human body temperature. The mutation also severely impacts the intramolecular mobility of calmodulin, especially in the apo form. Glycine 113 acts as an alpha-helical C-capping residue in both apo/ - and Ca 2+ /calmodulin, but its exchange to arginine has very different effects on the apo and Ca 2+ forms. The majority of arrhythmogenic calmodulin variants identified affects residues in the Ca 2+ coordinating loops of the two C-domain EF-Hands, causing a 'direct impact on Ca 2+ binding'. However, G113R lies outside a Ca 2+ coordinating loop and acts differently and more similar to the previously characterized arrhythmogenic N53I. Therefore, we suggest that altered apo/CaM dynamics may be a novel general disease mechanism, defining low-calcium target affinity - or Ca 2+ binding kinetics - critical for timely coordination of essential ion-channels in the excitation-contraction cycle., Competing Interests: Declaration of Competing Interest MTO acted as expert witness for the defense in the 2022/2023 Inquiry into the convictions of Kathlen Megan Folbigg (https://2022folbigginquiry.dcj.nsw.gov.au/). CVH, NMP and HI are employees of private companies., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

As one of the grain crop pathogenic fungi with the greatest impacts on agricultural economical as well as human health, an elaborate understanding of the life cycle and subsequent metabolome of Fusarium graminearum is of great interest. Throughout the lifetime of the fungus, it is known to produce a wide array of secondary metabolites, including polyketides. One of the F. graminearum polyketides which has remained a mystery until now has been elucidated in this work. Previously, it was suggested that the biosynthetic product of the PKS2 gene cluster was involved in active mycelial growth, the exact mechanism, however, remained unclear. In our work, disruption and overexpression of the PKS2 gene in F. graminearum enabled structural elucidation of a linear and a cyclic tetraketide with a double methyl group, named fugralin A and B, respectively. Further functional characterization showed that the compounds are not produced during infection, and that deletion and overexpression did not affect pathogenicity or visual growth. The compounds were shown to be volatile, which could point to possible functions that can be investigated further in future studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Severinsen, Westphal, Terp, Sørensen, Olsen, Bachleitner, Studt-Reinhold, Wimmer, Sondergaard and Sørensen.)

Missense variants in CALM genes encoding the Ca 2+ -binding protein calmodulin (CaM) cause severe cardiac arrhythmias. The disease mechanisms have been attributed to dysregulation of RyR2, for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) and/or Ca V 1.2, for Long-QT Syndrome (LQTS). Recently, a novel CALM2 variant, G114R, was identified in a mother and two of her four children, all of whom died suddenly while asleep at a young age. The G114R variant impairs closure of Ca V 1.2 and RyR2, consistent with a CPVT and/or mild LQTS phenotype. However, the children carrying the CALM2 G114R variant displayed a phenotype commonly observed with variants in Na V 1.5 , i.e., Brugada Syndrome (BrS) or LQT3, where death while asleep is a common feature. We therefore hypothesized that the G114R variant specifically would interfere with Na V 1.5 binding. Here, we demonstrate that CaM binding to the Na V 1.5 IQ-domain is severely impaired for two CaM variants G114R and G114W. The impact was most severe at low and intermediate Ca 2+ concentrations (up to 4 µM) resulting in more than a 50-fold reduction in Na V 1.5 binding affinity, and a smaller 1.5 to 11-fold reduction at high Ca 2+ concentrations (25-400 µM). In contrast, the arrhythmogenic CaM-N98S variant only induced a 1.5-fold reduction in Na V 1.5 binding and only at 4 µM Ca 2+ . A non-arrhythmogenic I10T variant in CaM did not impair Na V 1.5 IQ binding. These data suggest that the interaction between Na V 1.5 and CaM is decreased with certain CaM variants, which may alter the cardiac sodium current, I Na . Overall, these results suggest that the phenotypic spectrum of calmodulinopathies may likely expand to include BrS- and/or LQT3-like traits., Competing Interests: MN and MO acted as expert witnesses in the 2022 New South Wales Inquiry into the convictions of the Australian woman carrying the CaM-G114R variant. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brohus, Busuioc, Wimmer, Nyegaard and Overgaard.)

Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that causes a wide range of infections. Its resistance to β-lactam antibiotics complicates treatment due to the limited number of antibiotics with activity against MRSA. To investigate development of alternative therapeutics, the mechanisms that mediate antibiotic resistance in MRSA need to be fully understood. In this study, MRSA cells were subjected to antibiotic stress from methicillin in combination with three cannabinoid compounds and analyzed using proteomics to assess the changes in physiology. Subjecting MRSA to nonlethal levels of methicillin resulted in an increased production of penicillin-binding protein 2 (PBP2). Exposure to cannabinoids showed antibiotic activity against MRSA, and differential proteomics revealed reduced levels of proteins involved in the energy production as well as PBP2 when used in combination with methicillin.

Accurate and absolute quantification of peptides in complex mixtures using quantitative mass spectrometry (MS)-based methods requires foreground knowledge and isotopically labeled standards, thereby increasing analytical expenses, time consumption, and labor, thus limiting the number of peptides that can be accurately quantified. This originates from differential ionization efficiency between peptides and thus, understanding the physicochemical properties that influence the ionization and response in MS analysis is essential for developing less restrictive label-free quantitative methods. Here, we used equimolar peptide pool repository data to develop a deep learning model capable of identifying amino acids influencing the MS1 response. By using an encoder-decoder with an attention mechanism and correlating attention weights with amino acid physicochemical properties, we obtain insight on properties governing the peptide-level MS1 response within the datasets. While the problem cannot be described by one single set of amino acids and properties, distinct patterns were reproducibly obtained. Properties are grouped in three main categories related to peptide hydrophobicity, charge, and structural propensities. Moreover, our model can predict MS1 intensity output under defined conditions based solely on peptide sequence input. Using a refined training dataset, the model predicted log-transformed peptide MS1 intensities with an average error of 9.7 ± 0.5% based on 5-fold cross validation, and outperformed random forest and ridge regression models on both log-transformed and real scale data. This work demonstrates how deep learning can facilitate identification of physicochemical properties influencing peptide MS1 responses, but also illustrates how sequence-based response prediction and label-free peptide-level quantification may impact future workflows within quantitative proteomics., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors.)

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead ( Bothrops atrox ). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of Leishmania ( Leishmania ) amazonensis . Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Carbon monoxide (CO) is the leading cause of death by poisoning worldwide. The aim was to explore the effects of mild and severe poisoning on blood gas parameters and metabolites. Eleven pigs were exposed to CO intoxication and had blood collected before and during poisoning. Mild CO poisoning (carboxyhaemoglobin, COHb 35.2 ± 7.9%) was achieved at 32 ± 13 minutes, and severe poisoning (69.3 ± 10.2% COHb) at 64 ± 23 minutes from baseline (2.9 ± 0.5% COHb). Blood gas parameters and metabolites were measured on a blood gas analyser and nuclear magnetic resonance spectrometer, respectively. Unsupervised principal component, analysis of variance and Pearson's correlation tests were applied. A P-value ≤ .05 was considered statistically significant. Mild poisoning resulted in a 28.4% drop in oxyhaemoglobin (OHb) and 12-fold increase in COHb, while severe poisoning in a 65% drop in OHb and 24-fold increase in COHb. Among others, metabolites implicated in regulation of metabolic acidosis (lactate, P < .0001), energy balance (pyruvate, P < .0001; 3-hydroxybutyrc acid, P = .01), respiration (citrate, P = .007; succinate, P = .0003; fumarate, P < .0001), lipid metabolism (glycerol, P = .002; choline, P = .0002) and antioxidant-oxidant balance (glutathione, P = .03; hypoxanthine, P < .0001) were altered, especially during severe poisoning. Our study adds new insights into the deranged metabolism of CO poisoning and leads the way for further investigation., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Introduction: Chronic hepatitis B (CHB) affects 257 million individuals worldwide with an annual estimated mortality rate of 880,000 individuals. Accurate diagnosis of the stage of disease is difficult, and there is considerable uncertainty concerning the optimal point in time, when treatment should be started., Objectives: By analyzing and comparing the metabolomes of patients at different stages of CHB and comparing them to healthy individuals, we want to determine the metabolic signature of disease progression and develop a more accurate metabolome-based method for diagnosis of disease progression ultimately giving a better basis for treatment decisions., Methods: In this study, we used the combination of transient elastography and serum metabolomics of 307 serum samples from a group of 90 patients with CHB before and under treatment (with a follow-up time up to 10 years) at different progression stages over the clinical phases and 43 healthy controls.., Results: Our data show that the metabolomics approach can successfully discover CHB changing from the immune tolerance to the immune clearance phase and show distinctive metabolomes from different medical treatment stages. Perturbations in ammonia detoxification, glutamine and glutamate metabolism, methionine metabolism, dysregulation of branched-chain amino acids, and the tricarboxylic acid (TCA) cycle are the main factors involved in the progression of the disease. Fluctuations increasing in aspartate, glutamate, glutamine, methionine and 13 other metabolites are fingerprints of progression., Conclusions: The metabolomics approach may expand the diagnostic armamentarium for patients with CHB. This method can provide a more detailed decision basis for starting medical treatment.

Serum, urine and tissue from a rat model of chronic kidney disease (CKD) were analysed using nuclear magnetic resonance (NMR) spectroscopy-based metabolomics methods, and compared with samples from sham operated rats. Both urine and serum were sampled at multiple timepoints, and the results have been reported elsewhere (https://doi.org/10.1007/s11306-019-1569-3[1]). The data could be useful to researchers working with human CKD or rat models of the disease. In addition, several different types of NMR spectra were recorded, including 1D NOESY, CPMG, and 2D J-resolved spectra, and the data could be useful for method comparison and algorithm development, both in terms of NMR spectroscopy and multivariate analysis., Competing Interests: The NMR laboratory at Aalborg University is supported by the Obel Family, SparNord and Carlsberg Foundations. Animal experiments were carried out at the Water and Salt Research Centre, Aarhus University, which was established and supported by the Danish National Research Foundation., (© 2020 The Authors.)

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo - Bl LPMO10A from Bacillus licheniformis , along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15 N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63 Cu(II)-bound 15 N- Bl LPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x 2 -y 2 ) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O 2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O 2 activation in chitin-active AA10 LPMOs., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Cardiovascular disease is the leading cause of death worldwide and cardiac surgery is a key treatment. This study explores metabolite changes as a consequence of ischemia-reperfusion due to cardiac surgery with the use of cardiopulmonary bypass (CPB). To describe the ischemia-reperfusion injury, metabolite changes were monitored in fifty patients before and after CPB at multiple time points. We describe a longitudinal metabolite dataset containing nearly 600 serum nuclear magnetic resonance (NMR) spectra obtained from samples collected simultaneously from the pulmonary artery (deoxygenated blood) and left atrium (oxygenated blood) before ischemia (pre-CPB), immediately after reperfusion (end-CPB), and the following 2, 4, 8, and 20 hours postoperatively. In addition, a longitudinal dataset including 57 quantified metabolites is also provided. These datasets will help researchers studying ischemia-reperfusion injury, as well as the time-dependent alterations related to the surgical trauma and the subsequent processes required in regaining metabolite balance. The datasets could also be used for the development of processing algorithms for NMR-based metabolomics studies and methods for the analysis of longitudinal multivariate data.

Key Points: Mutations in the calmodulin protein (CaM) are associated with arrhythmia syndromes. This study focuses on understanding the structural characteristics of CaM disease mutants and their interactions with the voltage-gated calcium channel Ca V 1.2. Arrhythmia mutations in CaM can lead to loss of Ca 2+ binding, uncoupling of Ca 2+ binding cooperativity, misfolding of the EF-hands and altered affinity for the calcium channel. These results help us to understand how different CaM mutants have distinct effects on structure and interactions with protein targets to cause disease., Abstract: Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium sensing protein whose sequence is completely conserved across all vertebrates. These mutations have been shown to interfere with the function of cardiac ion channels, including the voltage-gated Ca 2+ channel Ca V 1.2 and the ryanodine receptor (RyR2), in a mutation-specific manner. The ability of different CaM disease mutations to discriminate between these channels has been enigmatic. We present crystal structures of several C-terminal lobe mutants and an N-terminal lobe mutant in complex with the Ca V 1.2 IQ domain, in conjunction with binding assays and complementary structural biology techniques. One mutation (D130G) causes a pathological conformation, with complete separation of EF-hands within the C-lobe and loss of Ca 2+ binding in EF-hand 4. Another variant (Q136P) has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca 2+ -saturating conditions when unbound to the IQ domain. Ca 2+ binding to a pair of EF-hands normally proceeds with very high cooperativity, but we found that N98S CaM can adopt different conformations with either one or two Ca 2+ ions bound to the C-lobe, possibly disrupting the cooperativity. An N-lobe variant (N54I), which causes severe stress-induced arrhythmia, does not show any major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of Ca V 1.2. These findings show that different CaM mutants have distinct effects on both the CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease., (© 2020 The Authors. The Journal of Physiology © 2020 The Physiological Society.)

Background: In our metabolomics studies we have noticed that repeated NMR acquisition on the same sample can result in altered metabolite signal intensities., Aims: To investigate the reproducibility of repeated NMR acquisition on selected metabolites in serum and plasma from two large human metabolomics studies., Methods: Two peak regions for each metabolite were integrated and changes occurring after reacquisition were correlated., Results: Integral changes were generally small, but serum citrate signals decreased significantly in some samples., Conclusions: Several metabolite integrals were not reproducible in some of the repeated spectra. Following established protocols, randomising analysis order and biomarker validation are important.

Background: Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation., Results: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens -mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II) 2 Cys 6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin., Conclusion: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin ( PKS3 : fsr ) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19 F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19 F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19 F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

Calmodulin (CaM) represents one of the most conserved proteins among eukaryotes and is known to bind and modulate more than a 100 targets. Recently, several disease-associated mutations have been identified in the CALM genes that are causative of severe cardiac arrhythmia syndromes. Although several mutations have been shown to affect the function of various cardiac ion channels, direct structural insights into any CaM disease mutation have been lacking. Here we report a crystallographic and NMR investigation of several disease mutant CaMs, linked to long-QT syndrome, in complex with the IQ domain of the cardiac voltage-gated calcium channel (Ca V 1.2). Surprisingly, two mutants (D95V, N97I) cause a major distortion of the C-terminal lobe, resulting in a pathological conformation not reported before. These structural changes result in altered interactions with the Ca V 1.2 IQ domain. Another mutation (N97S) reduces the affinity for Ca 2+ by introducing strain in EF hand 3. A fourth mutant (F141L) shows structural changes in the Ca 2+ -free state that increase the affinity for the IQ domain. These results thus show that different mechanisms underlie the ability of CaM disease mutations to affect Ca 2+ -dependent inactivation of the voltage-gated calcium channel., Competing Interests: The authors declare no conflict of interest.

NMR spectroscopy is a well-established technique to determine the structure of peptides and small proteins in solution, also when bound to detergent micelles or phospholipid bicelles. The structure of the peptide alone is, however, not conveying the full picture, if the peptide is bound to a micelle, since it does not tell anything about the orientation of the peptide in the micelle. This article describes how to obtain that information together with information on peptide structure.

The apo-form of the 23.3 kDa catalytic domain of the AA9 family lytic polysaccharide monooxygenase NcLPMO9C from Neurospora crassa has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the (1)H, (13)C, and (15)N chemical shift assignments of this LPMO.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 10(5) M(-1) ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad., (© 2014 The Protein Society.)

The 19.9 kDa C-terminal module (R3) from Azotobacter vinelandii mannronan C5-epimerase AlgE6 has been (13)C, (15)N isotopically labelled and recombinantly expressed. We report here the (1)H, (13)C, (15)N resonance assignment of AlgE6R3.

In this paper we describe a novel method for quantification of amino acids. First, α-hydroxy acid derivatives of amino acids were formed after reaction with dinitrogen trioxide by the van Slyke reaction. Second, the α-hydroxy acid derivatives were separated on an Aminex HPX-87H column (Bio-Rad) eluted isocratically with 5 mM H(2)SO(4) and quantified by refractive index detection. We were able to measure the reaction products of 13 of the 20 classical amino acids: glycine, l-alanine, l-valine, l-leucine, l-isoleucine, l-methionine, l-serine, l-threonine, l-asparagine, l-glutamine, l-aspartic acid, l-glutamic acid, and l-proline. We obtained linear relationships between the product peak areas and initial amino acid concentration, whereby the concentrations of these amino acids could be quantified on the basis of the quantification of their products. The method can be used to analyze amino acids in parallel with other small molecules, such as sugars or short chain fatty acids, and was used for parallel quantification of glycine, l-alanine, or l-glutamic acid, and glucose uptake in cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii . The method can also be used to quantify other amines, as demonstrated by detection of Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol).

Alginate epimerases are large multidomain proteins capable of epimerising C5 on beta-D-mannuronic acid (M) turning it into alpha-L-guluronic acid (G) in a polymeric alginate. Azotobacter vinelandii secretes a family of seven epimerases, each of which is capable of producing alginates with characteristic G distribution patterns. All seven epimerases consist of two types of modules, denoted A and R, in varying numbers. Attempts to study these enzymes with solution-state NMR are hampered by their size-the smallest epimerase, AlgE4, consisting of one A- and one R-module, is 58 kDa, resulting in heavy signal overlap impairing the interpretation of NMR spectra. Thus we obtained segmentally (2)H, (15)N labeled AlgE4 isotopomeres (A-[(2)H, (15)N]-R and [(2)H, (15)N]-A-R) by protein trans-splicing using the naturally split intein of Nostoc punctiforme. The NMR spectra of native AlgE4 and the ligated versions coincide well proving the conservation of protein structure. The activity of the ligated AlgE4 was verified by two different enzyme activity assays, demonstrating that ligated AlgE4 displays the same catalytic activity as wild-type AlgE4.

The murine 10-residue neurohormone kisspeptin (YNWNSFGLRY) is an important regulator of reproductive behavior and gonadotrophin secretion. It is known to form a random coil in solution, but undergoes a structural change in the presence of membranes although the nature of this change is not fully determined. The peptide's conformational versatility raises the question whether it is also able to form ordered aggregates under physiological conditions, which might be relevant as a storage mechanism. Here we show that heparin induces kisspeptin to form beta-sheet rich amyloid aggregates both at neutral (pH 7.0) and slightly acidic (pH 5.2) conditions. Addition of heparin leads to aggregation after a certain lag phase, irrespective of the time of addition of heparin, indicating that heparin is needed to facilitate the formation of fibrillation nuclei. Aggregation is completely inhibited by submicellar concentrations of zwitterionic and anionic surfactants. Unlike previous reports, our NMR data do not indicate persistent structure in the presence of zwitterionic surfactant micelles. Thus kisspeptin can aggregate under physiologically relevant conditions provided heparin is present, but the process is highly sensitive to the presence of amphiphiles, highlighting the very dynamic nature of the peptide conformation and suggesting that kisspeptin aggregation is a biologically regulatable process.

Three series of novel water-soluble beta-cyclodextrin-dextran polymers have been prepared by "click" chemistry. The polymers were synthesized from alkyne-modified dextrans (AMDs) onto which mono-6-O-deoxy-monoazido-betaCD (N3betaCD) was grafted by a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The polymers have been characterized by NMR spectroscopy and size exclusion chromatography (SEC). The binding properties have been characterized by isothermal titration calorimetry (ITC) and show excellent accessibility of the betaCDs.

A method called Dock'n'Flash was developed to offer site-specific capture and direct UVA-induced photocoupling of recombinant proteins. The method involves the tagging of recombinant proteins with photoreactive p-benzoyl-L-phenylalanine (pBpa) by genetic engineering. The photoreactive pBpa tag is used for affinity capture of the recombinant protein by beta-cyclodextrin (beta-CD), which provides hydrogen atoms to be abstracted in the photocoupling process. To exemplify the method, a recombinant, folded, and active N27pBpa mutant of cutinase from Fusarium solani pisi was produced in E. coli. Insertion of pBpa was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. A molecular dynamic simulation, with water as solvent, showed high solvent accessibility of the pBpa benzophenone group in N27pBpa-cutinase mutant. The formation of an inclusion complex between the benzophenone group of N27pBpa-cutinase and beta-CD was shown, and an apparent K(d) of 1.65 mM was determined using (1)H NMR. Photocoupling of beta-CD to N27pBpa-cutinase in a 1:1 ratio, upon UVA irradiation at 360 +/- 20 nm, was shown by MALDI-TOF mass spectroscopy. UVA photoimmobilization of N27pBpa-cutinase on quartz slides coated with beta-CD was achieved from liquid or dry films by total internal reflection fluorescence (TIRF). The Dock'n'Flash method offers a solution for direct photocoupling and patterning of recombinant proteins onto surfaces with site-specific attachment.

In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.

p25alpha is a 219-residue protein which stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25alpha has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25alpha is folded under physiological conditions, since it has a well structured and solvent-sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25alpha undergoes clear spectral changes suggesting significant loss of structure. p25alpha unfolds cooperatively in urea according to a simple two-state transition with a stability in water of approximately 5 kcal/mol. The protein behaves as a monomer and refolds with a transient on-pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25alpha lacking residues 3-43 also unfolds cooperatively and with similar stability, suggesting that the N-terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3-4 molecules of tubulin per p25alpha and is not significantly affected by the deletion of residues 3-43. In conclusion, we dismiss the proposal that p25alpha is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin-binding properties.

The purpose of this work was to study the inclusion binding interaction of two structurally related steroids, prednisolone and 6alpha-methyl prednisolone with a wide variety of cyclodextrins (CDs), both native (alpha-, beta- and gamma-CD) as well as modified (hydroxypropyl-, sulfobutyl ether-, glucosyl-, and maltosyl-beta-CD and sulfobutyl ether-gamma-CD), and to relate the binding constants to structural differences in the steroids. Phase-solubility diagrams of the steroids with various CDs were obtained. The stability constants (K1:1) revealed that beta-CD formed the strongest complex with prednisolone, followed by gamma-CD. With 6alpha-methyl prednisolone, the stability constants of both beta- and alpha-CD were considerably lower compared with prednisolone. In contrast, gamma-CD formed a stronger complex with 6alpha-methyl prednisolone compared with prednisolone. Derivatives of beta-CD gave slightly altered stability constants compared with native beta-CD. The structures of the complexes between the two guest molecules and native beta-CD were studied by nuclear magnetic resonance spectroscopy. This clearly showed that the introduction of the methyl group led to a different binding geometry of 6alpha-methyl prednisolone compared with prednisolone. Additionally, the nuclear magnetic resonance results indicate the presence of an additional, weaker binding site, which is less populated in prednisolone., (Copyright 2005 Wiley-Liss, Inc. and the American Pharmacists Association.)

We present a calorimetric investigation of stabilisation of hen egg-white lysozyme with sorbitol in the pH range 3.8-10.5. Differential scanning calorimetry and steady-state fluorescence were used to determine the denaturation temperatures of lysozyme as a function of sorbitol concentration. The fluorescence data were collected in the presence of 2M urea to lower the melting point of the protein to an observable range of the instrument. The effect of sorbitol on the activation energy of unfolding was investigated by scanrate studies. The effect of sorbitol lysozyme interaction was investigated using isothermal titration calorimetry. The titration experiments were performed with folded as well as unfolded lysozyme to investigate in more detail the nature of the interaction. The data obtained in those experiments show a remarkable stabilisation effect of sorbitol. We observed a 4.0 degrees C increase in the Tm for 1 M sorbitol in the pH range 3.8-8.5 by scanning calorimetry. The effect increases dramatically at pH 9.5 where we observe a 9.5 degrees C stabilisation. An increase in the sorbitol concentration to 2 M stabilises lysozyme by 11.3-13.4 degrees C in the pH range 9.5-10.5. In the absence of urea, no significant effects of sorbitol were observed on the activation energy for unfolding for lysozyme at pH 4.5. This indicates together with the results from the titration experiments that sorbitol may stabilise the folded form of lysozyme by destabilising the unfolded form of lysozyme. At pH values at and above lysozyme's pI (approximately 9.3), the unfolding of the protein is accompanied with a substantial amount of self-aggregation seen in the calorimetry experiments in the ratio of DeltaH(cal)/DeltaH(vH). In the presence of sorbitol, the self-aggregation was counterbalanced by higher sorbitol concentrations. These results strongly suggest a negative influence of sorbitol on the unfolded form of lysozyme and thereby stabilising the native form.

Many therapeutic proteins require storage at room temperature for extended periods of time. This can lead to aggregation and loss of function. Cyclodextrins (CDs) have been shown to function as aggregation suppressors for a wide range of proteins. Their potency is often ascribed to their affinity for aromatic amino acids, whose surface exposure would otherwise lead to protein association. However, no detailed structural studies are available. Here we investigate the interactions between human growth hormone (hGH) and different CDs at low pH. Although hGH aggregates readily at pH 2.5 in 1 M NaCl to form amorphous aggregates, the presence of 25 to 50 mM of various beta-CD derivatives is sufficient to completely avoid this. alpha- and gamma-CD are considerably less effective. Stopped-flow data on the aggregation reaction in the presence of beta-CD are analyzed according to a minimalist association model to yield an apparent hGH-beta-CD dissociation constant of approximately 6 mM. This value is very similar to that obtained by simple fluorescence-based titration of hGH with beta-CD. Nuclear magnetic resonance studies indicate that beta-CD leads to a more unfolded conformation of hGH at low pH and predominantly binds to the aromatic side-chains. This indicates that aromatic amino acids are important components of regions of residual structure that may form nuclei for aggregation.