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Aspergillus oryzae ß-D-galactosidase (ß-Gal) efficiently hydrolyzes sesaminol triglucoside into sesaminol, which has higher biological activity. However, ß-Gal is difficult to be separate from the reaction mixture and limited by stability. To resolve these problems, ß-Gal was immobilized on amino-functionalized magnetic nanoparticles mesoporous silica pre-activated with glutaraldehyde (Fe3O4@mSiO2-ß-Gal), which was used for the first time to prepare sesaminol. Under the optimal conditions, the immobilization yield and recovered activity of ß-Gal were 57.9 ± 0.3 % and 46.5 ± 0.9 %, and the enzymatic loading was 843 ± 21 Uenzyme/gsupport. The construction of Fe3O4@mSiO2-ß-Gal was confirmed by various characterization methods, and the results indicated it was suitable for heterogeneous enzyme-catalyzed reactions. Fe3O4@mSiO2-ß-Gal was readily separable under magnetic action and displayed improved activity in extreme pH and temperature conditions. After 45 days of storage at 4 °C, the activity of Fe3O4@mSiO2-ß-Gal remained at 92.3 ± 2.8 %, which was 1.29 times than that of free enzyme, and its activity remained above 85 % after 10 cycles. Fe3O4@mSiO2-ß-Gal displayed higher affinity and catalytic efficiency. The half-life was 1.41 longer than free enzymes at 55.0 °C. Fe3O4@mSiO2-ß-Gal was employed as a catalyst to prepare sesaminol, achieving a 96.7 % conversion yield of sesaminol. The excellent stability and catalytic efficiency provide broad benefits and potential for biocatalytic industry applications.
Assuntos
Aspergillus oryzae , Enzimas Imobilizadas , Glutaral , Dióxido de Silício , beta-Galactosidase , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Aspergillus oryzae/enzimologia , Dióxido de Silício/química , Glutaral/química , Dioxóis/química , Dioxóis/farmacologia , Nanopartículas de Magnetita/química , Porosidade , Temperatura , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , FuranosRESUMO
The development of peptide-based materials is one of the most challenging aspects of biomaterials research in recent years. The assembly of peptides is mainly controlled by forces such as hydrogen bonding, hydrophobic interaction, electrostatic interaction, and π-π accumulation. Peptides have unique advantages such as simple structure, easy synthesis, good biocompatibility, non-toxicity, easy modification, etc. These factors make peptides turn into ideal biomedical materials, and they have a broad application prospect in biomedical materials, and thus have received wide attention. In this review, the mechanism and classification of peptide self-assembly and its applications in biomedicine and hydrogels were introduced.
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Materiais Biocompatíveis , Hidrogéis , Peptídeos , Humanos , Materiais Biocompatíveis/química , Hidrogéis/química , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/química , Eletricidade Estática , AnimaisRESUMO
Based on the typical similar repeat units (abcdefg)n of α-helical structure, the peptide H was designed to self-assemble into an organohydrogel in response to pH. Depending on the different pH, the proportions of secondary structure, microstructure, and mechanical properties of the gel were investigated. Circular dichroism (CD) and Fourier transform infrared (FT-IR) showed that the proportion of α-helical structure gradually increased to become dominant with the increase of pH. Combining transmission electron microscopy (TEM) and atomic force microscopy (AFM), it was found that the increase of the ordered α-helix structure promoted fiber formation. The further increase in pH changed the intermolecular forces, resulting in an increase in the α-helix content and the enhancement of helix-helix interaction, causing the gel fibers to converge into thicker and more dense ones. The temperature test showed the stable rheological properties of the organohydrogel between 20-60 °C. Drug release and cytotoxicity showed that the DOX-loaded organohydrogel could have a better release in an acidic environment, indicating its potential application as a drug local delivery carrier.
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Retinal segmentation is a crucial step in the early warning of human health conditions. However, retinal blood vessels possess complex curvature, irregular distribution, and contain multi-scale fine structures, which make the limited receptive field of regular convolution challenging to process their vascular details efficiently. Additionally, the encoder-decoder based network leads to irreversible spatial information loss because of multiple downsampling, resulting in over-segmentation and missed segmentation of the vessels. For this reason, we develop a high-resolution network based on Deformable Convolution v3, called HRD-Net. By constructing a high-resolution representation, the network allows special attention to be paid to the details of tiny blood vessels. The proposed feature enhancement cascade module based on Deformable Convolution v3 can flexibly adapt and capture the ever-changing morphology and intricate connections of retinal blood vessels, ensuring the continuity of vessel segmentation. In the output phase of the network, the proposed global aggregation module integrates full-resolution feature maps while suppressing redundant features, achieving an effective fusion of high-level semantic information and spatial detail information. In addition, we have re-examined the selection criteria for activation and normalization methods, and also refine the network architectures from a spatial domain perspective to release redundant computational loads. Testing on the DRIVE, STARE, and CHASE_DB1 datasets indicates that HRD-Net, with fewer parameters, outperforms existing segmentation methods on several evaluation metrics such as F1, ACC, SE, SP, AUC, and IOU.
Assuntos
Aprendizagem , Vasos Retinianos , Humanos , Vasos Retinianos/diagnóstico por imagem , Benchmarking , Retina/diagnóstico por imagem , Salários e Benefícios , Processamento de Imagem Assistida por Computador , AlgoritmosRESUMO
Cecropin A (1-7) is a cationic antimicrobial peptide which contain lots of basic amino acids. To understand the effect of basic amino acids on cecropin A (1-7), analogues CA2, CA3 and CA4 which have more arginine or lysine at the N-terminal or C-terminal were designed and synthesized. The interaction of cecropin A (1-7) and its analogs with DNA was studied using ultraviolet-visible spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Multispectral analysis showed that basic amino acids improved the interaction between the analogues and DNA. The interaction between CA4 and DNA is most pronounced. Fluorescence spectrum indicated that Ksv value of CA4 is 1.19 × 105 L mol-1 compared to original peptide cecropin A (1-7) of 3.73 × 104 L mol-1. The results of antimicrobial experiments with cecropin A (1-7) and its analogues showed that basic amino acids enhanced the antimicrobial effect of the analogues. The antimicrobial activity of CA4 against E. coli was eightfold higher than that of cecropin A (1-7). The importance of basic amino acid in peptides is revealed and provides useful information for subsequent studies of antimicrobial peptides.
Assuntos
Dicroísmo Circular , DNA , Escherichia coli , Escherichia coli/efeitos dos fármacos , DNA/química , DNA/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Testes de Sensibilidade MicrobianaRESUMO
Phase separation, a biophysical segregation of subcellular milieus referred as condensates, is known to regulate transcription, but its impacts on physiological processes are less clear. Here, we demonstrate the formation of liquid-like nuclear condensates by SGF29, a component of the SAGA transcriptional coactivator complex, during cellular senescence in human mesenchymal progenitor cells (hMPCs) and fibroblasts. The Arg 207 within the intrinsically disordered region is identified as the key amino acid residue for SGF29 to form phase separation. Through epigenomic and transcriptomic analysis, our data indicated that both condensate formation and H3K4me3 binding of SGF29 are essential for establishing its precise chromatin location, recruiting transcriptional factors and co-activators to target specific genomic loci, and initiating the expression of genes associated with senescence, such as CDKN1A. The formation of SGF29 condensates alone, however, may not be sufficient to drive H3K4me3 binding or achieve transactivation functions. Our study establishes a link between phase separation and aging regulation, highlighting nuclear condensates as a functional unit that facilitate shaping transcriptional landscapes in aging.
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Self-assembled peptide-based hydrogels have shown great potential in bio-related applications due to their porous structure, strong mechanical stability, high biocompatibility, and easy functionalization. Herein, the structure and characteristics of hydrogels and the mechanism of action of several regular secondary structures during gelation are investigated. The factors influencing the formation of peptide hydrogels, especially the pH responsiveness and salt ion induction are analyzed and summarized. Finally, the biomedical applications of peptide hydrogels, such as bone tissue engineering, cell culture, antigen presentation, antibacterial materials, and drug delivery are reviewed.
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Hidrogéis , Peptídeos , Hidrogéis/química , Peptídeos/química , Sistemas de Liberação de Medicamentos , Antibacterianos/química , Técnicas de Cultura de CélulasRESUMO
Hair loss affects millions of people at some time in their life, and safe and efficient treatments for hair loss are a significant unmet medical need. We report that topical delivery of quercetin (Que) stimulates resting hair follicles to grow with rapid follicular keratinocyte proliferation and replenishes perifollicular microvasculature in mice. We construct dynamic single-cell transcriptome landscape over the course of hair regrowth and find that Que treatment stimulates the differentiation trajectory in the hair follicles and induces an angiogenic signature in dermal endothelial cells by activating HIF-1α in endothelial cells. Skin administration of a HIF-1α agonist partially recapitulates the pro-angiogenesis and hair-growing effects of Que. Together, these findings provide a molecular understanding for the efficacy of Que in hair regrowth, which underscores the translational potential of targeting the hair follicle niche as a strategy for regenerative medicine, and suggest a route of pharmacological intervention that may promote hair regrowth.
Assuntos
Células Endoteliais , Quercetina , Camundongos , Animais , Quercetina/farmacologia , Cabelo , Folículo Piloso , AlopeciaRESUMO
Precise dissection of DNA-protein interactions is essential for elucidating the recognition basis, dynamics and gene regulation mechanism. However, global profiling of weak and dynamic DNA-protein interactions remains a long-standing challenge. Here, we establish the light-induced lysine (K) enabled crosslinking (LIKE-XL) strategy for spatiotemporal and global profiling of DNA-protein interactions. Harnessing unique abilities to capture weak and transient DNA-protein interactions, we demonstrate that LIKE-XL enables the discovery of low-affinity transcription-factor/DNA interactions via sequence-specific DNA baits, determining the binding sites for transcription factors that have been previously unknown. More importantly, we successfully decipher the dynamics of the transcription factor subproteome in response to drug treatment in a time-resolved manner, and find downstream target transcription factors from drug perturbations, providing insight into their dynamic transcriptional networks. The LIKE-XL strategy offers a complementary method to expand the DNA-protein profiling toolbox and map accurate DNA-protein interactomes that were previously inaccessible via non-covalent strategies, for better understanding of protein function in health and disease.
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DNA , Fatores de Transcrição , Fatores de Transcrição/química , DNA/química , Aminas/química , Ligação Proteica , Reagentes de Ligações Cruzadas/químicaRESUMO
Various peptide drugs have entered the market with the development of molecular biology. Peptide drugs are used for treat diseases such as diabetes, breast cancer, and HIV infection. In this study, three nicotinamide-modified peptides were synthesized by modifying the N-terminus of BRCA1 (856-871, Y856R, K862Y, R866W) peptide with three nicotinic acid derivatives using solid-phase peptide synthesis. The results of calf thymus DNA (ctDNA) binding activity indicated that binding constants of BRCA1 (856-871, Y856R, K862Y, R866W) (P0) and three nicotinamide-modified peptides (P1, P2, and P3) to ctDNA were 1.89 × 103, 2.97 × 104, 7.61 × 104, and 8.09 × 104 L·mol-1, respectively. The binding affinity of the modified peptides was superior to that of BRCA1 (856-871, Y856R, K862Y, R866W). ΔHθ < 0 and ΔSθ < 0 indicated that van der Waals force and hydrogen bond contributed most to peptide-ctDNA binding. Results obtained by Circular dichroism (CD) indicated that peptide binding interaction led to conformational changes in ctDNA. Ultraviolet-visible (UV) spectroscopy, ethidium bromide (EB) competition experiments, DNA melting experiments, and viscosity measurements verified that peptides interacted with ctDNA via groove binding. Ionic strength experiments manifested that electrostatic binding was also involved in peptide-ctDNA binding.
Assuntos
Infecções por HIV , Niacinamida , Humanos , Termodinâmica , Dicroísmo Circular , Peptídeos , Espectrometria de Fluorescência/métodos , Simulação de Acoplamento Molecular , Espectrofotometria Ultravioleta , Proteína BRCA1RESUMO
As a bridge of human-computer communication, the color design of intelligent vehicle HMI interactive interface is particularly important. It is also the first guide to the driver during the driving process. The quality of its design will also directly affect the driver's senses and the driving safety of the vehicle. Therefore, this paper introduces the current situation, design principle, and future development of the vehicle interaction interface from multiple perspectives. Through the neural network system (condition generation countermeasure network model) of visual recognition, the color of the intelligent vehicle HMI interactive interface under the user experience is analyzed. According to the analysis of the psychological cognition and behavior operation of the automobile user, the correlation analysis of the human, vehicle, environment, and various elements of the interface is carried out, and how the vehicle interactive interface can meet the expected physiological and psychological needs of the user more and improve the operability is discussed in order to design an on-board HMI interactive interface that can be intelligently perceived according to weather, driver's interests, and other factors and then improve the current backward operation mode of the on-board interactive interface, so that the interaction between people and vehicles is more smooth and pleasant.
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Condução de Veículo , Percepção de Cores , Humanos , Condução de Veículo/psicologia , Automóveis , Redes Neurais de ComputaçãoRESUMO
Peptide drugs, which are mainly used for the treatment of AIDS, myeloma, and breast cancer, have evolved rapidly owing to their high efficacy and low side effects. The interaction mechanisms of two peptide drugs with two biological macromolecules (protein and DNA), which are of great significance in disease prevention and drug design, were investigated using molecular docking, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, UV-visible spectroscopy and viscosity measurements. The interaction between a series of common drugs and ovalbumin (OVA) was simulated by molecular docking, and two peptide drugs with the highest energy values, namely atazanavir and carfilzomib, were selected; the binding energy values of these drugs with OVA were -59.20 and -55.93 kcal/mol, respectively. The Kb values of the interaction of the two drugs with OVA/DNA were in the range of 104-107 M-1, and the binding affinity of the drugs was stronger with OVA than with DNA. Hydrogen bonds and van der Waals forces were very important for the binding between drugs and OVA through molecular docking studies, and it was consistent with experimental results (ΔH < 0, ΔH < 0). The synchronous fluorescence spectrum showed that the interaction caused a change to the original structure of OVA, and atazanavir had a greater effect on OVA than carfilzomib. CD spectrum analysis also demonstrated that the conformation of OVA changed slightly. The interaction between atazanavir and DNA was mainly driven by hydrophobic forces (ΔH > 0 and ΔH > 0), whereas the major interaction forces involved in the binding of carfilzomib with DNA were hydrogen bonds and van der Waals forces. DNA melting studies, UV-visible spectroscopy, CD spectroscopy and viscosity measurements established that the interaction between the drugs and DNA was groove binding.
Assuntos
Peptídeos , Preparações Farmacêuticas , Sítios de Ligação , Dicroísmo Circular , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Espectrometria de Fluorescência , TermodinâmicaRESUMO
We propose that the teratoma, a recognized standard for validating pluripotency in stem cells, could be a promising platform for studying human developmental processes. Performing single-cell RNA sequencing (RNA-seq) of 179,632 cells across 23 teratomas from 4 cell lines, we found that teratomas reproducibly contain approximately 20 cell types across all 3 germ layers, that inter-teratoma cell type heterogeneity is comparable with organoid systems, and teratoma gut and brain cell types correspond well to similar fetal cell types. Furthermore, cellular barcoding confirmed that injected stem cells robustly engraft and contribute to all lineages. Using pooled CRISPR-Cas9 knockout screens, we showed that teratomas can enable simultaneous assaying of the effects of genetic perturbations across all germ layers. Additionally, we demonstrated that teratomas can be sculpted molecularly via microRNA (miRNA)-regulated suicide gene expression to enrich for specific tissues. Taken together, teratomas are a promising platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.
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Linhagem da Célula , Modelos Biológicos , Teratoma/patologia , Animais , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Teratoma/genéticaRESUMO
BRCA2 is an important tumor suppressor gene that plays a critical role in preserving the stability of cellular genetic information, participating in DNA repair by engaging in binding interactions with RAD51 proteins. However, the lack of structural data on BRCA2 and RAD51 makes the study of their interaction mechanism still a great challenge. We characterize the structure of the BRC8-RAD51 complex using ZDOCK protein docking software and identify the potential non-conserved active site of BRC8 via virtual alanine scanning, utilizing the obtained results to synthesize BRC8, its six analogous peptides (BRC8-1 to BRC8-6), and critical peptide fragment of RAD51 (RAD51(231-260)) by Fmoc solid-phase synthesis. The analogous peptides are found to exhibit a secondary structure significantly different from that of BRC8 by circular dichroism spectroscopy, which indicates that mutation sites determined by computer-aided simulation correspond to key amino acid residues substantially affecting polypeptide structure. On the other hand, the secondary structure of RAD51(231-260) was also considerably influenced by its interaction with BRC8 and analogs, e.g., the fraction of the α-helical structure in RAD51(231-260) increased to 23.6, 15.1, and 13.5% upon interaction with BRC8-1, BRC8-3, and BRC8-6, respectively. The results show that the properties of C-terminal amino acid residues significantly influence peptide-peptide interactions, in agreement with the results of virtual alanine scanning. Therefore, computer-aided simulation was confirmed to be a technique that is useful for narrowing down the range of sites responsible for interactions between peptides or proteins, and provides new inspirations for the design of peptides with strong interactions.
Assuntos
Proteína BRCA2/química , Desenho de Fármacos , Fragmentos de Peptídeos/química , Rad51 Recombinase/química , Proteína BRCA2/metabolismo , Humanos , Conformação Proteica , Rad51 Recombinase/metabolismoRESUMO
For the first time, the [4 + 3] or [2 + 1] annulation of crotonate-derived sulfur ylides with arylidenemalononitrile or arylidene-1H-indene-1,3(2H)-dione is reported using Na2CO3 as the base. This protocol is advantageous as it does not require prior preparation of arylidenemalononitrile or arylidene-1H-indene-1,3(2H)-dione substrates, due to the independent participation of the base in the two reactions. This mild, operationally multicomponent process can be employed for the transformation of a wide variety of commercially available aldehydes into the corresponding indeno[1,2-b]oxepine or cyclopropyl acrylate core in moderate to excellent yields under mild conditions.
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Polypeptides with finger-like structures can often intercalate into the grooves of DNA, thereby affecting DNA repair or activating gene transcription, both of which are crucial for the regulation of physiological processes. Their conserved amino acid sequence and simple structure have provided useful elements for the design and assembly of functional molecules. In this paper, using the C2H2 zinc finger domain and the PEP-FOLD3 online simulation platform 11 polypeptides containing 22 amino acid residues were designed. In addition, the CD spectroscopy was combined with the fluorescence spectroscopy to study the polypeptide structures and their interaction with DNA. Results showed that although addition of zinc ions affected the polypeptide structure, particularly of the polypeptides A4, B1, and B3, zinc ion was not an essential factor for increasing polypeptide-DNA interactions. Our study revealed an increase in the interaction strength between mutated polypeptides and DNA, suggesting that mutations disrupt polypeptide structure, and polypeptides interact with DNA by groove and electrostatic binding. Mutations at the 12th and 15th amino acid residues had the greatest effect. The stronger binding between A2 or B2 and DNA indicates that the polypeptide has a spatial structure that can stably interact with DNA. The structure and characteristics of these polypeptide domains can provide information for the design and development of new polypeptide functional molecules, which could have potential significance and applications. However, this information also suggests that there are many challenges facing polypeptide design due to the synergistic effects between the side chains of amino acid residues.
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Dedos de Zinco CYS2-HIS2/genética , Peptídeos , Engenharia de Proteínas/métodos , Software , Sequência de Aminoácidos , Sequência Conservada/genética , DNA/química , DNA/metabolismo , Modelos Moleculares , Mutação/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Dobramento de ProteínaRESUMO
Understanding the effects of genetic perturbations on the cellular state has been challenging using traditional pooled screens, which typically rely on the delivery of a single perturbation per cell and unidimensional phenotypic readouts. Here, we use barcoded open reading frame overexpression libraries coupled with single-cell RNA sequencing to assay cell state and fitness, a technique we call SEUSS (scalable functional screening by sequencing). Using SEUSS, we perturbed hPSCs with a library of developmentally critical transcription factors (TFs) and assayed the impact of TF overexpression on fitness and transcriptomic states. We further leveraged the versatility of the ORF library approach to assay mutant genes and whole gene families. From the transcriptomic responses, we built genetic co-regulatory networks to identify altered gene modules and found that KLF4 and SNAI2 drive opposing effects along the epithelial-mesenchymal transition axis. From the fitness responses, we identified ETV2 as a driver of reprogramming toward an endothelial-like state.
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Reprogramação Celular/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Linhagem Celular , Transição Epitelial-Mesenquimal , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Análise de Sequência de RNA , Análise de Célula Única , Fatores de Transcrição/metabolismoRESUMO
Zinc finger protein ZNF191(243-368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243-368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243-368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His6-tag system could be used to express ZNF191(243-368). The presence of the His6-tag at the N-terminus of ZNF191(243-368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His6-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.
Assuntos
Fatores de Transcrição Kruppel-Like , Mutação , Proteínas Recombinantes de Fusão , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Mutations in breast cancer susceptibility gene 2 (BRCA2) can lead to chromosomal instability and result in breast cancer, which is strongly associated with p53 mutations. Here, based on the crystal structure of BRC4 and p53, the spatial structure of BRC2 and p53 (171-192) was simulated, providing structural basis for the site-specific mutation of BRC2. The BRC analogous peptides and p53 (171-192) were synthesized, and the interaction between the mutant peptide and p53 (171-192) was studied using circular diachronic spectroscopy and fluorescence spectroscopy. The results show that the mutations of amino acid residues constituting the BRC2 α-helix significantly affect the structure and interaction of BRC analogs and p53 (171-192), which provides support for understanding the structure of the BRC repeat motifs and its interaction pattern with p53.