Umami Polypeptide Detection System Targeting The Human T1R1 Receptor And Its Taste-Presenting Mechanism

As one of the basic tastes of human taste perception,umami is very important in the human perception system.Umami receptors can recognize a variety of umami substances such as LGlu,nucleotides,organic acids,and larger oligopeptides.Among them,the recognition of LGlu by glutamate-metabolized umami receptors is relatively clear.However,T1R1/T1R3 umami receptors are difficult to express and purify,and their structures are not resolved,resulting in unclear ligand characteristics and mechanisms,which severely limits the development of umami.At present,it has been determined that the extracellular end of the human T1R1 receptor(hT1R1-VFT)is responsible for the recognition and binding of MSG,but whether it has an affinity for umami peptides and its binding characteristics are still unclear.Therefore,in this study,two recombinant protein heterologous expression systems were used to express T1R1 receptor ectoproteins to obtain a large number of recombinant proteins,and to establish targeting T1R1 receptors based on protein-ligand interaction detection methods and computational simulation methods.The umami peptide detection system was developed to explore the kinetics of binding of different umami peptides to the hT1R1 receptor and explain the umami mechanism of umami peptides.The main research contents are as follows:(1)Mammalian cell expression system and E.coli expression system for constructing hT1R1-VFT protein.In the mammalian cell expression system,after HEK293F cell culture and target gene transfection,Western-Blot verified that the target protein expression level was very small,which could not meet the requirements of the protein amount required for the further study of affinity with the polypeptides.In the E.coli expression system,the T1R1-VFT target protein induced and expressed was analyzed by polyacrylamide gel electrophoresis,and it was found that the target protein existed in the form of inclusion bodies,and the inclusion bodies were denatured and renatured.The protein was gradually dialyzed,and the concentration of the denaturant was slowly reduced,and the target T1R1-VFT protein with high purity and protein amount meeting the experimental requirements was successfully obtained.Finally,the renatured T1R1-VFT protein was analyzed by circular dichroism,and it was confirmed that the protein obtained by the E.coli expression system was the target protein with physiological functions.(2)Based on the expressed hT1R1-VFT target protein,a new umami polypeptide detection system targeting human T1R1 receptor was established by using different ligand-receptor interaction detection methods.Firstly,four kinds of umami peptides were synthesized,and the affinity of peptides with T1R1-VFT was detected by fluorescence quenching spectrometry.The Ka value of GRVSNCAA was 479.55 M-1,KGGGGP was 108.56 M-1,while that of MSG was 32.97 M-1,so the two peptides had stronger affinity to hT1R1-VFT than MSG.Although the Ka values of KGDEESLA and TGDPEK are relatively low,which are 3.82 M-1 and 2.67 M-1 respectively,it still shows that the umami peptide can interact with hT1R1-VFT.Therefore,all the four peptides can be proved to have affinity with hT1R1-VFT by fluorescence quenching spectroscopy,and the affinity of MSG to T1R1-VFT is in the middle level relative to the peptides.Surface plasmon resonance and isothermal titration calorimetry were used to verify the affinity between fresh peptides and T1R1-VFT.Model fitting further showed that octapeptide GRVSNCAA had strong affinity with T1R1-VFT,and the other three peptides also interacted with hT1R1-VFT.This study shows that umami peptides have a strong affinity for human T1R1,and for the first time,the conjecture of polypeptides targeting T1R1 receptors is supported by experimental data,and a polypeptide detection system targeting human T1R1 receptors is established.(3)After determining the interaction between umami peptides and T1R1-VFT,the interaction force and binding characteristics of the peptides were further analyzed by computational simulation method.Homologous modeling was carried out by using the analyzed structure of the same family,and then molecular docking was carried out after evaluating the rationality of the model,followed by MD simulation and binding free energy calculation,and then found out the binding law of T1R1 receptor and medium-long chain umami peptide,which laid the foundation for the next design of new high-strength umami peptide.Through MD simulation and binding free energy analysis,and combined with the study of the properties of peptides,it was found that the hydrophobic peptides showed higher affinity receptor characteristics and lower binding free energy with receptors,indicating that umami peptides and T1R1-VFT receptors formed a more affinity complex,that is,umami peptides had higher umami intensity and lower threshold for sensory evaluation.Therefore,in the process of screening new umami peptides,the appropriate hydrophobic amino acid residues are worth considering.In this study,through heterologous expression of hT1R1-VFT protein,the binding mechanism of umami peptides to T1R1 receptors was deeply studied by means of ligandreceptor affinity detection and computational simulation,and a umami peptides detection system targeting human T1R1 receptors was successfully established.Our results verify the high affinity of umami peptides to hT1R1,and the affinity data of the four peptides and the trend of sensory evaluation of taste threshold are the same,so it is considered that receptor T1R1 is the main affinity receptor of umami taste peptides,while MSG can stimulate T1R1 at the same time in addition to the traditional binding glutamate metabolic receptor,and the stimulation intensity is in the middle level.Computational simulations show that the fact that peptides occupy different binding sites in the hT1R1 binding pocket due to conformational changes is an important factor for showing different taste thresholds,and the hydrophobicity of peptides plays an important role in the affinity process.The system established in this paper makes it possible to quickly screen high-strength umami peptides and taste detection sensors based on T1R1 receptors,which is helpful to the design of umami peptides with high umami ability.