Analysis of enzyme-responsive peptide surfaces by Raman spectroscopy.
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ABSTRACT:
SUBMITTER: Sahoo JK
PROVIDER: S-EPMC4819759 | biostudies-literature | 2016 Mar
REPOSITORIES: biostudies-literature
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Project description:BackgroundChemistry and particularly enzymology at surfaces is a topic of rapidly growing interest, both in terms of its role in biological systems and its application in biocatalysis. Existing protein immobilization approaches, including noncovalent or covalent attachments to solid supports, have difficulties in controlling protein orientation, reducing nonspecific absorption and preventing protein denaturation. New strategies for enzyme immobilization are needed that allow the precise control over orientation and position and thereby provide optimized activity.Methodology/principal findingsA method is presented for utilizing peptide ligands to immobilize enzymes on surfaces with improved enzyme activity and stability. The appropriate peptide ligands have been rapidly selected from high-density arrays and when desirable, the peptide sequences were further optimized by single-point variant screening to enhance both the affinity and activity of the bound enzyme. For proof of concept, the peptides that bound to ?-galactosidase and optimized its activity were covalently attached to surfaces for the purpose of capturing target enzymes. Compared to conventional methods, enzymes immobilized on peptide-modified surfaces exhibited higher specific activity and stability, as well as controlled protein orientation.Conclusions/significanceA simple method for immobilizing enzymes through specific interactions with peptides anchored on surfaces has been developed. This approach will be applicable to the immobilization of a wide variety of enzymes on surfaces with optimized orientation, location and performance, and provides a potential mechanism for the patterned self-assembly of multiple enzymes on surfaces.
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Project description:The ever-growing demand for new drugs highlights the need to develop novel cost- and time-effective techniques for drug discovery. Surface-enhanced Raman spectroscopy (SERS) is an emerging ultrasensitive and label-free technique that allows for the efficient detection and characterization of molecular interactions. We have recently developed a SERS platform for detecting a single protein molecule linked to a gold substrate (Almehmadi et al. Scientific Reports 2019). In this study, we extended the approach to probe the binding of potential drugs to RNA targets. To demonstrate the proof of concept, two 16-amino acid residue peptides with close primary structures and different binding affinities to the RNA CUG repeat related to myotonic dystrophy were tested. Three-microliter solutions of the RNA repeat with these peptides at nanomolar concentrations were probed using the developed approach, and the binding of only one peptide was demonstrated. The SER spectra exhibited significant fluctuations along with a sudden strong enhancement as spectra were collected consecutively from individual spots. Principal component analysis (PCA) of the SER spectral datasets indicated that free RNA repeats could be differentiated from those complexed with a peptide with 100% accuracy. The developed SERS platform provides a novel opportunity for label-free screening of RNA-binding peptides for drug discovery. Schematic representation of the SERS platform for drug discovery developed in this study.
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Project description:Chlorpyrifos (CPF) and 2,4-dichlorophenoxyacetic acid (2,4-D) are insecticides and herbicides which has been widely used on farms. However, CPF and 2,4-D residues on corps can bring high risks to human health. Accurate detection of pesticide residues is important for controlling health risks caused by CPF and 2,4-D. Therefore, we developed a fast, sensitive, economical, and lossless surface-enhanced Raman spectroscopy (SERS)-based method for pesticide detection. It can rapidly and simultaneously determine the CPF and 2,4-D mixed pesticide residues on an apple surface at a minimum of 0.001 mg L-1 concentration, which is far below the pesticide residue standard in China and the EU. The limits of detection reach down to 1.28 × 10-9 mol L-1 for CPF and 2.47 × 10-10 mol L-1 for 2,4-D. The limits of quantification are 4.27 × 10-9 mol L-1 and 8.23 × 10-10 mol L-1 for CPF and 2,4-D. This method has a great potential for the accurate detection of pesticide residues, and may be applied to other fields of agricultural products and food industry.
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Project description:Hydration is ubiquitous in any kind of water-substance interaction such as in various interfacial and biological processes. Despite substantial progress made to date, however, still less explored is the hydration behavior on complex heterogeneous surfaces, such as the water surrounding the protein, which requires a platform that enables systematic investigation at the atomic scale. Here, we realized a heterogeneous self-assembled monolayer system that allows both controllable mixing with hydrophobic or hydrophilic groups and precise distance control of the functional carboxyl groups from the surface by methylene spacer groups. Using surface-enhanced Raman spectroscopy (SERS), we first demonstrated the hydrophobic (or hydrophilic) mixing ratio-dependent pK a variation of the carboxyl group. Interestingly, we observed a counterintuitive, non-monotonic behavior that a fractionally mixed hydrophobic group can induce significant enhancement of dielectric strength of the interfacial water. In particular, such a fractional mixing substantially decreases the amide coupling efficiency at the surface, as manifested by the corresponding pK a decrease. The SERS-based platform we demonstrated can be widely applied for atomically precise control and molecular-level characterization of hydration water on various heterogeneous surfaces of biological and industrial importance.
| S-EPMC7706083 | biostudies-literature
Project description:For enzyme-catalysed biotransformations, continuous in situ detection methods minimise the need for sample manipulation, ultimately leading to more accurate real-time kinetic determinations of substrate(s) and product(s). We have established for the first time an on-line, real-time quantitative approach to monitor simultaneously multiple biotransformations based on UV resonance Raman (UVRR) spectroscopy. To exemplify the generality and versatility of this approach, multiple substrates and enzyme systems were used involving nitrile hydratase (NHase) and xanthine oxidase (XO), both of which are of industrial and biological significance, and incorporate multistep enzymatic conversions. Multivariate data analysis of the UVRR spectra, involving multivariate curve resolution-alternating least squares (MCR-ALS), was employed to effect absolute quantification of substrate(s) and product(s); repeated benchmarking of UVRR combined with MCR-ALS by HPLC confirmed excellent reproducibility.
| S-EPMC5488198 | biostudies-literature
Project description:Peptide surfaces were obtained by the covalent immobilisation of fluorescently labelled pentapeptides carboxyfluorescein-glycine-arginine-methionine-leucine-glycine, either directly or through a poly(ethylene glycol) (PEG) linker on modified silicon wafers. Each step during the preparation of the peptide surfaces was confirmed by several surface characterisation techniques. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy were used to determine the surface composition, the wafers philicity was measured by contact angle and atomic force microscopy was used to investigate the surface morphology. Exposure of the peptide surfaces to trypsin resulted in the release of a fluorescently labelled peptide product, which allowed the kinetics of the enzymatic reaction to be followed with the aid of fluorescence spectroscopy. The electrospray ionisation mass spectrometry analysis of the post-digestion solution confirmed that the pentapeptides attached to the solid support undergo specific trypsin hydrolysis at the C-terminus of the arginine residues. Detailed surface analyses before and after the enzyme action was performed using ToF-SIMS. Because of the limited accessibility of the short peptide directly attached to the surface, a quantitative yield of enzymatic hydrolysis was observed only in case when the peptide was bound through the PEG linker. The insertion of the PEG linker increased the number of immobilised peptides and the rate of enzymatic digestion which consequently improved the quality of the enzyme assays. The described approach may be used for different peptide sequences designed for other proteases.
| S-EPMC3825591 | biostudies-literature
Project description:We report a combined molecular dynamics (MD) and ab initio simulation study of the ultrafast broadband ultraviolet (UV) stimulated resonance Raman (SRR) spectra of the Trp-cage mini protein. Characteristic two dimensional (2D) SRR features of various folding states are identified. Structural fluctuations erode the cross peaks and the correlation between diagonal peaks is a good indicator of the ?-helix formation.
| S-EPMC3859311 | biostudies-literature
Project description:Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems.
| S-EPMC6385126 | biostudies-literature
Project description:Background and study aims
Bowel cancer is one of the commonest cancers worldwide. Earlier detection causes better outcomes for patients and longer survival. Symptoms of bowel cancer are non-specific and are shared by harmless bowel disorders. It is a challenge for doctors in general practice to diagnose bowel cancer and many symptomatic patients are sent to hospital for tests to rule it out. This is normally a colonoscopy, which is expensive, uncomfortable and can be harmful. The current approach to diagnosis causes great anxiety in patients waiting for these tests and is not a prudent approach to diagnosis. To allow earlier diagnosis by GPs we have studied whether a newly designed blood test taken in primary care is accurate and effective in patients with bowel symptoms that could be linked with cancer. This would benefit a large number of patients who are concerned about their bowel symptoms without the need for referral to hospital. It could also lead to diagnosis of bowel cancer at an earlier stage improving survival in the longer term. The aim of this study is to use a newly developed blood test for bowel cancer in primary care to achieve an earlier diagnosis. This would allow more timely appropriate treatment both for patients diagnosed with bowel cancer and those who are cancer free.
Who can participate?
Adults aged 50 years and older who have symptoms of bowel cancer.
What does the study involve?
Participants are randomly allocated to one of two groups. Those in the first group have their Raman spectroscopy blood test done immediately and those in the second grou have their sample tested after they’ve had a diagnosis. The amount of participants who are referred on the USC pathway from each group are assessed after 12 months.
| 2416539 | ecrin-mdr-crc
Project description:Self-assembled peptide nanostructures recently have gained much attention as drug delivery systems. As biomolecules, peptides have enhanced biocompatibility and biodegradability compared to polymer-based carriers. We introduce a peptide nanoparticle system containing arginine, histidine, and an enzyme-responsive core of repeating GLFG oligopeptides. GLFG oligopeptides exhibit specific sensitivity towards the enzyme cathepsin B that helps effective controlled release of cargo molecules in the cytoplasm. Arginine can induce cell penetration, and histidine facilitates lysosomal escape by its buffering capacity. Herein, we propose an enzyme-responsive amphiphilic peptide delivery system (Arg-His-(Gly-Phe-Lue-Gly)3, RH-(GFLG)3). The self-assembled RH-(GFLG)3 globular nanoparticle structure exhibited a positive charge and formulation stability for 35 days. Nile Red-tagged RH-(GFLG)3 nanoparticles showed good cellular uptake compared to the non-enzyme-responsive control groups with d-form peptides (LD (LRH-D(GFLG)3), DL (DRH-L(GFLG)3), and DD (DRH-D(GFLG)3). The RH-(GFLG)3 nanoparticles showed negligible cytotoxicity in HeLa cells and human RBCs. To determine the drug delivery efficacy, we introduced the anticancer drug doxorubicin (Dox) in the RH-(GFLG)3 nanoparticle system. LL-Dox exhibited formulation stability, maintaining the physical properties of the nanostructure, as well as a robust anticancer effect in HeLa cells compared to DD-Dox. These results indicate that the enzyme-sensitive RH-(GFLG)3 peptide nanoparticles are promising candidates as drug delivery carriers for biomedical applications.
| S-EPMC8779831 | biostudies-literature