ROS, continuously produced in cells, can reversibly or irreversibly oxidize proteins, lipids, and DNA. At the protein level, cysteine, methionine, tryptophan, and tyrosine residues are particularly prone to oxidation. Here, we describe the solid phase synthesis of peptides containing four different oxidation products of tryptophan residues that can be formed by oxidation in proteins in vitro and in vivo: 5-HTP, Oia, Kyn, and NFK. First, we synthesized Oia and NFK by selective oxidation of tryptophan and then protected the α-amino group of both amino acids, and the commercially available 5-HTP, with Fmoc-succinimide. High yields of Fmoc-Kyn were obtained by acid hydrolysis of Fmoc-NFK. All four Fmoc derivatives were successfully incorporated, at high yields, into three different peptide sequences from skeletal muscle actin, creatin kinase (M-type), and β-enolase. The correct structure of all modified peptides was confirmed by tandem mass spectrometry. Interestingly, isobaric peptides containing 5-HTP and Oia were always well separated in an acetonitrile gradient with TFA as the ion-pair reagent on a C₁₈-phase. Such synthetic peptides should prove useful in future studies to distinguish isobaric oxidation products of tryptophan.
To demonstrate the utility of the coarse-grained united-residue (UNRES) force field to compare experimental and computed kinetic data for folding proteins, we have performed long-time millisecondtimescale canonical Langevin molecular dynamics simulations of the triple β-strand from the Formin binding protein 28 WW domain and six nonnatural variants, using UNRES. The results have been compared with available experimental data in both a qualitative and a quantitative manner. Complexities of the folding pathways, which cannot be determined experimentally, were revealed. The folding mechanisms obtained from the simulated folding kinetics are in agreement with experimental results, with a few discrepancies for which we have accounted. The origins of single-and double-exponential kinetics and their correlations with two-and three-state folding scenarios are shown to be related to the relative barrier heights between the various states. The rate constants obtained from time profiles of the fractions of the native, intermediate, and unfolded structures, and the kinetic equations fitted to them, correlate with the experimental values; however, they are about three orders of magnitude larger than the experimental ones for most of the systems. These differences are in agreement with the timescale extension derived by scaling down the friction of water and averaging out the fast degrees of freedom when passing from all-atom to a coarse-grained representation. Our results indicate that the UNRES force field can provide accurate predictions of folding kinetics of these WW domains, often used as models for the study of the mechanisms of proein folding.FBP28 WW domain | nonnatural variants | folding rates | free-energy landscapes | millisecond-timescale canonical MD simulations R ecent advances in computer simulation techniques have facilitated the direct study of the folding process of small fastfolding proteins, using all-atom force fields (1). However, it is important to validate the simulation methodologies, and the only way to accomplish this is a quantitative comparison with experimental data with proper statistics. The validation of all-atom simulation methodologies is still a major problem because of the differences between the experimental timescale (from multiple microseconds to seconds) and the theoretical one (from hundreds of nanoseconds to microseconds). To overcome this problem, many approximate coarse-grained methods have been developed during the past decade (2-5). One of them makes use of a physics-based united-residue (UNRES) force field developed in our group over the past years (6-14) (SI Appendix, Fig. S1 and SI Materials and Methods).The folding and unfolding rates are among the most accessible quantitative observables for two-and multistate folding proteins; therefore, a study of protein folding kinetics can bridge microscopic motions and the world of experimental measurements. In analyzing protein folding kinetics, the differential rate equations and their integrated forms become more complex as the num...
The term reactive oxygen species refers to small molecules that can oxidize, for example, nearby proteins, especially cysteine, methionine, tryptophan, and tyrosine residues. Tryptophan oxidation is always irreversible in the cell and can yield several oxidation products, such as 5-hydroxy-tryptophan (5-HTP), oxindolylalanine (Oia), kynurenine (Kyn), and N-formyl-kynurenine (NFK). Because of the severe effects that oxidized tryptophan residues can have on proteins, there is a great need to develop generally applicable and highly sensitive techniques to identify the oxidized residue and the oxidation product. Here, the fragmentation behavior of synthetic peptides corresponding to sequences recently identified in three skeletal muscle proteins as containing oxidized tryptophan residues were studied using postsource decay and collision-induced dissociation (CID) in matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry (MS) and CID in an electrospray ionization (ESI) double quadrupole TOF-MS. For each sequence, a panel of five different peptides containing Trp, 5-HTP, Kyn, NFK, or Oia residue was studied. It was always possible to identify the modified positions by the y-series and also to distinguish the different oxidation products by characteristic fragment ions in the lower mass range by tandem MS. NFK- and Kyn-containing peptides displayed an intense signal at m/z 174.1, which could be useful in identifying accordingly modified peptides by a sensitive precursor ion scan. Most importantly, it was always possible to distinguish isomeric 5-HTP and Oia residues. In ESI- and MALDI-MS/MS, this was achieved by the signal intensity ratios of two signals obtained at m/z 130.1 and 146.1. In addition, high collision energy CID in the MALDI-TOF/TOF-MS also permitted the identification of these two isomeric residues by their v- and w-ions, respectively.
Random peptide libraries that cover large search spaces are often used for the discovery of new binders, even when the target is unknown. To ensure an accurate population representation, there is a tendency to use large libraries. However, parameters such as the synthesis scale, the number of library members, the sequence deconvolution and peptide structure elucidation, are challenging when increasing the library size. To tackle these challenges, we propose an algorithm-supported approach to peptide library design based on molecular mass and amino acid diversity. The aim is to simplify the tedious permutation identification in complex mixtures, when mass spectrometry is used, by avoiding mass redundancy. For this purpose, we applied multi (two- and three-)-objective genetic algorithms to discriminate between library members based on defined parameters. The optimizations led to diverse random libraries by maximizing the number of amino acid permutations and minimizing the mass and/or sequence overlapping. The algorithm-suggested designs offer to the user a choice of appropriate compromise solutions depending on the experimental needs. This implies that diversity rather than library size is the key element when designing peptide libraries for the discovery of potential novel biologically active peptides. Electronic supplementary material The online version of this article (10.1186/s13321-019-0347-6) contains supplementary material, which is available to authorized users.
Passing through the blood-brain barrier (BBB) to treat neurological conditions is one of the main hurdles in modern medicine. Many drugs with promising in vitro profiles become ineffective in vivo due to BBB restrictive permeability. In particular, this includes drugs such as antiviral porphyrins, with the ability to fight brain-resident viruses causing diseases such as HIV-associated neurocognitive disorders (HAND). In the last two decades, BBB shuttles, particularly peptide-based ones, have shown promise in carrying various payloads across the BBB. Thus, peptide−drug conjugates (PDCs) formed by covalent attachment of a BBB peptide shuttle and an antiviral drug may become key therapeutic tools in treating neurological disorders of viral origin. In this study, we have used various approaches (guanidinium, phosphonium, and carbodiimide-based couplings) for on-resin synthesis of new peptide−porphyrin conjugates (PPCs) with BBB-crossing and potential antiviral activity. After careful fine-tuning of the synthetic chemistry, DIC/oxyma has emerged as a preferred method, by which 14 different PPCs have been made and satisfactorily characterized. The PPCs are prepared by coupling a porphyrin carboxyl group to an amino group (either N-terminal or a Lys side chain) of the peptide shuttle and show effective in vitro BBB translocation ability, low cytotoxicity toward mouse brain endothelial cells, and low hemolytic activity. Three of the PPCs, MP-P5, P4-MP, and P4-L-MP, effectively inhibiting HIV infectivity in vitro, stand out as most promising. Their efficacy against other brain-targeting viruses (Dengue, Zika, and SARS-CoV-2) is currently under evaluation, with preliminary results confirming that PPCs are a promising strategy to treat viral brain infections.
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