Protein Disulfide Isomerase and Its Potential Function on Endoplasmic Reticulum Quality Control in Diatom Phaeodactylum tricornutum

PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under differentstresses. However, bioinformatic characteristics and potential functions of PDIs in diatom Phaeodactylumtricornutum (Pt) are still unknown so far. Hence, the genome-wide characteristics of PtPDI proteins in P. tricornutumwere first studied via bioinformatic and transcriptomic methods. 42 PtPDI genes were identified from thegenome of P. tricornutum. The motif, protein structure, classification, number of introns, phylogenetic relationship,and the expression level of 42 PtPDI genes under the tunicamycin stress were analyzed. A pair of tandemduplicated genes (PtPDI15 and PtPDI18) was observed in P. tricornutum. The 42 PtPDIs with different genecharacteristics were divided into three independent clades, indicating different evolutional relationships and functionsof these PtPDIs. The 14 up-regulated PtPDI genes under the tunicamycin treatment might have a positiveeffect on the ER quality control of the unfolded/misfolded proteins, while the 7 down-regulated PtPDIs mightnegatively affect the ERQC. The characteristics of all 42 PtPDIs and their proposed working model here providea comprehensive understanding of the PtPDIs gene family. The differential expression of 21 PtPDIs will be usefulfor further functional study in the ERQC.

Protein Disulfide Isomerase A2 Is Correlated with Immune Infiltrates and Is a Novel Prognostic Biomarker in Glioma Patients

Objective Protein disulfide isomerase A2(PDIA2),a member of the protein disulfide isomerase family,plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds,together with enzymes such as thiol isomerase,oxidase,and reductase.This study investigated the clinical significance and potential functions of PDIA2 in glioma.Methods The expression of PDIA2 in gliomas was explored using The Cancer Genome Atlas and Gene Expression Omnibus databases.We analyzed the clinical characteristics of glioma patients and the prognostic and diagnostic value of PDIA2 expression.Kaplan-Meier and Cox regression analyses were used to examine the effect of PDIA2 expression on overall survival,progression-free interval,and disease-specific survival.Furthermore,we performed Gene Set Enrichment Analysis and immune infiltration analysis to investigate the functions of PDIA2.PDIA2 mRNA and protein expression was evaluated in cell lines and glioma tissues.Results PDIA2 was expressed at low levels in glioma patients.Kaplan-Meier survival analysis showed that glioma patients with low PDIA2 levels had a worse prognosis than those with high PDIA2 levels.Receiver operating characteristic curve analysis indicated the diagnostic and prognostic ability of PDIA2(area under the curve=0.918).Pathways associated with PD1,PI3K/AKT,cancer immunotherapy via PD1 blockade,Fceri-mediated NF-kB activation,FOXM1,and DNA repair were enriched in glioma patients with low levels of PDIA2.PDIA2 expression levels were negatively correlated with immune cell infiltrate levels.Conclusion PDIA2 levels are significantly downregulated in glioma.PDIA2 expression may be a potential biomarker for the diagnosis and prognosis of glioma patients.

Bioinformatics Analysis and Homology Modeling Study of Protein Disulfide Isomerase(mPDI) from Medicago sativa L.

pdi gene from Medicago sativa L. ,encoding Protein Disulfide Isomerase（ mPDI ）, has been cloned and sequenced. According to the mRNA and amino acid sequence, the character of mPDI such as the physical and chemical properties, hydrophilicity/hydrophobicity, signal peptide, secondary structure, coiled coil, transmembrane domains, O-glycogylation site, active site, subcellular localization, functional structural domains and three-dimensional structure were analyzed by a series of bioinformatics software. The results showed that mPDI was a hydrophobic and stable protein with 3 coiled coils, 30-glycogylation sites, 2 structural domains of thioredoxin, 2 active sites of thioredoxin, and located in rough endoplasmic reticulum. It has 512 amino acids, the theoretical pl is 4.98, and signal peptide located in 1-24AA. In the secondary structure, a-helix, random coil, extended chain is 26.37%, 53.32%, 20.31% respectively. The validation of modeling accords with the stereochemistry.

Prediction Method of Protein Disulfide Bond Based on Pattern Selection

The effect of the different training samples is different for the classifier when pattern recognition system is established. The training samples were selected randomly in the past protein disulfide bond prediction methods, therefore the prediction accuracy of protein contact was reduced. In order to improve the influence of training samples, a prediction method of protein disulfide bond on the basis of pattern selection and Radical Basis Function neural network has been brought forward in this paper. The attributes related with protein disulfide bond are extracted and coded in the method and pattern selection is used to select training samples from coded samples in order to improve the precision of protein disulfide bond prediction. 200 proteins with disulfide bond structure from the PDB database are encoded according to the encoding approach and are taken as models of training samples. Then samples are taken on the pattern selection based on the nearest neighbor algorithm and corresponding prediction models are set by using RBF neural network. The simulation experiment result indicates that this method of pattern selection can improve the prediction accuracy of protein disulfide bond.

Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia co/i and its “oxidizing” mutant

AIM: To study the influence of redox environment of Escherichia coli (E(?) coli) cytoplasm on disulfide bond formation of recombinant proteins. METHODS: Bovine fibrobllast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E(?)coli strains BL21(DE3) and M15[pREP4] respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain,E(?)coli Origami(DE3). The 4 recombinant E(?)coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively. RESULTS: All recombinant E(?)coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv] was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from inclusion body in M15[pQE-HBscFv] was 30-35 mg/L and the affinity constant was 1.98×107 mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains. CONCLUSION: Modification of the redox environment of E(?)coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

Quantification of Global Protein Disulfides and Thiol-Protein Mixed Disulfides to Study the Protein Dethiolation Mechanisms

The redox state of cellular thiols is widely studied because it was recently linked to many different diseases and pathologies. In this work we quantified the concentrations of protein disulfides (PSSP) and thiol-protein mixed disulfides (XSSP) in rat tissues (liver, kidney and heart) and cells (Raw 264.7) by an improved method of XSSP and PSSP determination after oxidative stress induced by diamide. Under native and denaturing conditions, a thiol block by N-ethymaleimide was introduced to avoid thiol exchange reaction activations by protein SH groups (PSH) (PSH + XSSP ←→ PSSP + XSH) and alterations of original XSSP/PSSP levels. Low molecular weight thiols (XSH) and PSH were respectively measured by HPLC on supernatants and on corresponding pellets by DTNB (Ellman’s reagent) after dithiothreitol reduction. PSSP concentrations of liver, heart and kidney were respectively 0.304, 0.605 and 0.785 μmoles/g and after diamide exposure they were significantly augmented of about 65%-70% in liver and heart, but not in the kidney. Normal XSSP, that were -20 times lower than normal PSSP were induced by diamide in liver and heart of about 40 times, but not in kidney. Thermodynamic criteria regarding the pKa values of thiols engaged as PSSP and GSSP were used to interpret dethiolation mechanisms via thiol exchange reactions.

Evidence That Protein Disulfide Isomerase in Yeast Saccharomyces cerevisiae Is Transported from the ER to the Golgi Apparatus

Newly synthesized membrane and secretory proteins in cells undergo folding in the endoplasmic reticulum with the introduction of disulfide bonds and acquire the correct three-dimensional structure. Disulfide bonds are especially important for protein folding. It has been thought that formation of protein disulfide bonds in eukaryotes is mainly carried out by an enzyme called protein disulfide isomerase. Proteins, bearing the C-terminus of amino acids sequences with His-Asp-Glu-Leu (HDEL) sequence in yeast, in the endoplasmic reticulum (ER), which is a eukaryotic cellular organelle involved in protein synthesis, processing, and transport, have been considered to recycle between ER and Golgi apparatus. The proposal for this recycling model derives from the study of an HDEL-tagged fusion protein. Here, the localization and oligosaccharide modification of protein disulfide isomerase were investigated in yeast, and showed the first direct evidence that this intrinsic ER protein transports from ER to Golgi. Results suggest that this native protein is also accessible to post-ER enzymes, and yet accumulates in the ER.

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins.Improving the yield in K.marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering.To address these issues,linear and circular yeast artificial chromosomes of K.marxianus(KmYACs)were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K.marxianus.These modules contained up to seven genes with a maximum size of 15 kb.KmYACs carried telomeres either from K.marxianus or Tetrahymena.KmYACs were transferred successfully into K.marxianus and stably propagated without affecting the normal growth of the host,regardless of the type of telomeres and configurations of KmYACs.KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins.In high-density fermentation,the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l,the highest reported level to date in K.marxianus.Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis,enhanced flux entering the tricarboxylic acid cycle,and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins.Consistently,supplementing lysine or arginine further improved the yield.Therefore,KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research.Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins,and this strategy may be applied to optimize other microbial cell factories.

In Silico Disulfide Bond Engineering to Improve Human LEPTIN Stability

Enhancing the stability of biomolecules is one of the hot topics in industry.In this study,we enhanced the stability of an important protein called LEPTIN.LEPTIN is a hormone secreted by fat cells playing an essential role in body weight and composition,and its deficiency can result in several disorders.The treatment of related LEPTIN dysfunctions is often available in the form of injection.To decrease the cost and the frequency of its applications can be achieved by increasing its lifetime through engineering LEPTIN.In this study,to engineer LEPTIN,we have introduced disulfide bonds.Disulfide By Design server was used to predict the suitable nominate pairs,which suggested three pairs of amino acids to be mutated to cysteine for disulfide bond formation.Additionally,to further evaluate the effect of combined mutations,we combined these three nominated pairs to produce three more mutants.In order to assess the effect of introduced mutations,molecular dynamic(MD)simulation was performed.The result suggests that Mutant-1 is more stable in comparison to wild-type and the other mutants.Moreover,docking results showed that the introduced mutation does not affect the receptor binding performance;therefore,it can be considered a suitable choice for future protein engineering.

二硫键异构酶结构及生物学功能研究进展

蛋白质二硫键异构酶具有酶和分子伴侣的生物学活性,在蛋白质合成、分泌的过程中具有重要作用。本文对其分子结构、生物学功能及其在血栓性疾病、肿瘤、神经退化性疾病发生发展中的功能机制进行了综述。

小麦面筋蛋白遗传特性综合评价

本研究对44个小麦品种的19个面筋蛋白性状进行分析,运用主成分分析、相关性分析、隶属函数法和逐步回归等方法对面筋蛋白品质进行综合评价。结果表明:二硫键含量变幅为3.28~6.78μmol/g,巯基含量的变幅为13.30~16.66μmol/g。面筋蛋白性状变异范围在4.74%~89.46%,其中稳定时间、粉质质量指数等变异系数较大,表明蛋白质量性状的变异潜力较丰富。主成分分析将19个蛋白性状转换为3个综合因子,贡献率分别为40.80%、19.86%和15.88%,累计贡献率达到76.54%,可以很好地将面筋蛋白归为质量性状、聚集性状和数量性状3类。利用隶属函数法计算面筋蛋白综合评价值(D值),主成分因子中载荷较大的12个性状的平均值与D值均呈显著正相关。通过逐步回归建立了7个性状(二硫键含量、麦谷蛋白大聚体含量、蛋白质含量、干面筋含量、面筋指数、拉伸面积和最大峰值时间)作为自变量的回归方程。面包品质评价指标与D值之间具有较好的线性关系,决定系数R2在0.5614~0.7713之间,验证了D值的准确性和可行性。结论:不同筋力小麦面筋蛋白存在较丰富的变异潜力;采用多元统计分析方法综合评价面筋蛋白品质具有可行性;面筋蛋白的量与质以及面筋网络中二硫键的含量可作为小麦品质评价和改良的关键指标。

ATG5和PDIA3在宫颈癌组织中的表达及其临床意义

目的:探究免疫原性细胞死亡相关基因——自噬相关基因5(ATG5)和蛋白质二硫键异构酶A3(PDIA3)在宫颈癌组织中的表达及其与患者临床病理特征和预后的相关性、相关信号通路及药物敏感性。方法:收集自2016年1月至2023年12月间青岛大学附属医院60例宫颈癌癌组织标本作为实验组,26例因子宫肌瘤、子宫腺肌症切除的正常宫颈组织标本作为对照组,并收集相应的临床资料。采用免疫组化法检测宫颈癌组织和正常宫颈组织中ATG5、PDIA3蛋白的表达差异,分析其表达与各项临床病理参数之间的关系。基于基因表达水平值的交互式分析平台(GEPIA)在线分析宫颈癌组织中ATG5、PDIA3的表达水平对患者预后的影响,使用基因本体论(GO)、京都基因与基因组百科全书(KEGG)、基因集富集分析(GSEA)探究ATG5和PDIA3基因可能涉及的生物学功能及信号通路,用p RRophetic包分析宫颈癌患者癌组织中ATG5、PDIA3高低表达与患者对化疗药物的敏感性。结果:ATG5、PDIA3在宫颈癌组织中的表达率均显著高于正常宫颈组织(83.3%vs 11.5%,χ^(2)=39.538,P=0.001;75.0%vs 46.2%,χ^(2)=6.753,P=0.009),ATG5的表达水平在肿瘤直径、FIGO分期、淋巴结转移方面的差异显著(均P<0.01);PDIA3的表达水平在肿瘤直径、分化程度、FIGO分期和淋巴结转移方面的差异显著(P<0.05或P<0.01)。ATG5和PDIA3的表达水平呈正相关(r=0.679,P<0.001)。GEPIA在线分析网站预后分析显示,ATG5和PDIA3高表达宫颈癌患者的预后差(均P<0.05)。ATG5和PDIA3主要富集的功能及信号通路包括细胞增殖与分化、抗原加工与提呈、P53结合、Wnt信号通路、MAPK信号通路及mTOR信号通路。结论:ATG5和PDIA3在宫颈癌组织中呈高表达,两者高表达与患者不良预后有关,ATG5和PDIA3参与细胞增殖与分化、抗原加工提呈及多种信号通路,有望成为宫颈癌治疗的潜在靶点。

蛋白质二硫键异构酶结构与相关疾病研究进展

蛋白质二硫键异构酶(Protein disulfide isomerase,PDI)属于硫氧还蛋白家族,其分子质量为57 kD,主要存在于内质网中,且结构中有两个活性位点,在蛋白质折叠过程中催化二硫键的裂解、形成和重排。PDI活性异常会导致未折叠或错误蛋白的积累,引起内质网应激(ERs),从而导致未折叠蛋白反应(UPR)。研究表明:PDI与癌症、神经紊乱等疾病显著相关,PDI的表达水平升高与多种癌症的发生、发展相关。通过对PDI的蛋白结构、功能、活性检测及相关疾病进行综述,以期为PDI及其病理研究提供新思路。

Stress protein expression in early phase spinal cord ischemia/reperfusion injury

Spinal cord ischemia/reperfusion injury is a stress injury to the spinal cord. Our previous studies using differential proteomics identified 21 differentially expressed proteins （n 〉 2） in rabbits with spinal cord ischemia/reperfusion injury. Of these proteins, stress-related proteins included protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70. In this study, we established New Zealand rabbit models of spinal cord ischemia/reperfusion injury by abdominal aorta occlusion. Results demonstrated that hind limb function initially improved after spinal cord ischemia/reperfusion injury, but then deteriorated. The pathological morphology of the spinal cord became aggravated, but lessened 24 hours after reperfusion. However, the numbers of motor neurons and interneurons in the spinal cord gradually decreased. The expression of protein disulfide isomerase A3, stress-induced-phosphoprotein 1 and heat shock cognate protein 70 was induced by ischemia/reperfusion injury. The expression of these proteins increased within 12 hours after reperfusion, and then decreased, reached a minimum at 24 hours, but subsequently increased again to similar levels seen at 6-12 hours, showing a characterization of induction-inhibition-induc- tion. These three proteins were expressed only in cytoplasm but not in the nuclei. Moreover, the expression was higher in interneurons than in motor neurons, and the survival rate of interneurons was greater than that of motor neurons. It is assumed that the expression of stress-related proteins exhibited a protective effect on neurons.

Evolutionary Relationship of Wheat Protein Disulphide Isomerase (PDI) Gene Promoter Sequence Based on Phylogenetic Analysis

Protein disulphide isomerase (PDI) is an oxidoreductase enzyme abundant in the endoplasmic reticulum (ER). In plants, PDIs have been shown to assist the folding and deposition of seed storage proteins during the biogenesis of protein bodies in the endosperm. Cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv. Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species were reported in our previous publications. Promoter sequences of three homoeologous genes encoding typical PDI, located on chromosome group 4 of bread wheat, and PDI promoter sequence analysis of Triticum urartu, Aegilops speltoides and Aegilops tauschii had also been reported previously. In this study, we report the isolation and sequencing of a ~700 bp region, comprising ~600 bp of the putative promoter region and 88 bp of the first exon of the typical PDI gene, in five accessions each from Triticum urartu (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD). Sequence analysis indicated large variation among sequences belonging to the different genomes, while close similarity was found within each species and with the corresponding homoeologous PDI sequences of Triticum aestivum cv. CS (AABBDD) resulting in an overall high conservation of the sequence conferring endosperm-specific expression.

热诱导-自组装大豆蛋白凝胶结构与功能特性构效关系研究

目的研究不同11S/7S比例大豆蛋白热诱导凝胶结构与功能特性的关系。方法配比不同11S/7S比例蛋白分散液,在100℃下对其热诱导凝胶化,对其蛋白二级结构、Zeta电位、粒径、表面疏水性、浊度、凝胶流变学、质构学指标进行分析。结果随着11S/7S蛋白比例从0.5/1.0增加到3.0/1.0,蛋白凝胶的粒径增大到原来的1.91倍、二硫键含量从3.463μmol/g增大到6.551μmol/g(1.81倍)、表面疏水性减少了1/5。二级结构中β-折叠增加、α-螺旋减少以及Zeta电位绝对值的增加均与11S所占比例呈正相关;流变学频率扫描结果显示11S蛋白占比越高,凝胶的储能模量G′值越高,凝胶硬度也显著增加了43.2%。凝胶硬度与二硫键含量、表面疏水性的相关性系数绝对值分别是0.946和0.789。结论大豆蛋白热诱导凝胶中,11S蛋白比例增多,更多的二硫键形成促进了蛋白结构发生重排,疏水基团进一步掩埋形成紧密的凝胶网络结构。蛋白凝胶巯基及二硫键含量变化代表凝胶结构状态改变,二硫键含量越多,凝胶网络结构越紧密。

植物乳杆菌植酸酶理性设计及表达鉴定

植酸酶能将植酸水解成肌醇衍生物,并释放出无机磷酸,是一种重要的生物催化剂。植酸酶的热稳定性和比活性是决定其潜在应用价值的2个关键因素。该研究从植物乳杆菌中克隆到了具有233个氨基酸残基、高活性的植酸酶基因(LpPHY233),并在大肠杆菌中实现了高水平的异源表达,其表达水平是植物乳杆菌的800倍。基于理性设计的结果,通过二硫键工程显著提高了Lp PHY233的温度特性和催化性能。突变体Lp PHY233S58C/K61C的最适反应温度较Lp PHY233提高了15℃。此外,它还具有良好的pH特性和动力学参数,在食品加工和饲料添加剂领域具有较大的应用前景。该研究结果为植酸酶的进一步分子修饰和工业应用提供了坚实的理论基础。

脱乙酰魔芋葡甘聚糖对猪肉肌原纤维蛋白结构及凝胶特性的影响

研究添加不同质量分数(0%、0.125%、0.25%、0.5%和1%)的脱乙酰魔芋葡甘聚糖(deacetylated konjac glucomannan,DKGM)对猪肉肌原纤维蛋白(myofibrillar protein,MP)结构和凝胶性能的影响,通过分析其凝胶强度、保水性、水分分布、微观结构和凝胶分子力的变化,探究DKGM对MP凝胶特性的影响机制。结果表明,MP凝胶强度随着DKGM质量分数的增加而增大,添加量为0.25%时达到峰值,是对照组的1.55倍;DKGM的加入可以减缓水的流动性,从而提高凝胶持水力;冷场扫描电子显微镜观察发现添加DKGM可以促进凝胶形成更为均匀致密的网络结构;结构和作用力分析表明适量添加DKGM可以促进MP分子的展开和疏水基团的暴露,增加活性巯基的含量,诱导更多α-螺旋向β-折叠转变,增强MP凝胶的疏水相互作用和二硫键,从而改善MP凝胶强度和持水力。

分泌型蛋白质二硫键异构酶在肿瘤发生发展中的研究进展

蛋白质二硫键异构酶(PDIs)家族是一组主要定位在内质网的巯基二硫键氧化还原酶,PDIs参与蛋白质二硫键的形成、还原和异构化,在维持蛋白质结构稳定和内质网稳态中发挥重要作用。PDIs蛋白在细胞中除可定位于内质网中,还可分泌至细胞外,存在于细胞外基质以及循环血液中。PDIs可以由肿瘤细胞或者其他细胞直接分泌至肿瘤微环境中,影响肿瘤细胞的增殖和迁移。本文就分泌型PDIs的结构功能及其与肿瘤发生发展之间的关系进行综述。

谷朊抗冻多肽对面团冷冻保护作用研究

采用旋转流动仪、SDS-PAGE凝胶电泳和原位拉曼光谱等手段探究谷朊抗冻多肽对面团流变特性、蛋白分子质量分布和分子结构的影响规律。结果表明,经过冷冻(冻融和冻藏)处理后,与对照相比,加入谷朊抗冻多肽的面团黏弹性有所下降,谷朊抗冻多肽可以抑制冷冻过程中GMP的解聚。谷朊抗冻多肽的添加有效维持了面筋蛋白的二级结构和分子排列的有序性,与对照相比,在经过反复冻融和恒温冻藏处理后,α-螺旋结构的质量分数分别提高了3.1%、2.5%,二硫键g-g-g构型质量分数分别提高了8.8%、9.8%,同时使得氨基酸侧链中I 850/I 830值下降、I 760值提高。当谷朊抗冻多肽的质量分数为0.5%时最有效地抑制了冷冻过程中冰晶生长对面筋蛋白网络结构的破坏,谷朊抗冻多肽保护了面筋网络结构的完整性,维持了面筋蛋白在面团中的骨架作用。