Cover Feature: Superior Proton-Transfer Catalytic Promiscuity of Cytochrome c in Self-Organized Media (7/2021)

Evolutionarily elderly proteins commonly feature greater catalytic promiscuity. Cytochrome c is among the first set of proteins in evolution to have known prospects in electron transport and peroxidative properties. Here, we report that cyt c is also a proficient proton-transfer catalyst and enhances the Kemp elimination (KE; model reaction to show proton transfer catalytic property) by ∼750-fold on self-organized systems like micelles and vesicles. The self-organized systems mimic the mitochondrial environment in vitro for cyt c. Using an array of biophysical and biochemical mutational assays, both acid–base and redox mechanistic pathways have been explored. The histidine moiety close to hemin group (His18) is mainly responsible for proton abstraction to promote the concerted E2 pathway for KE catalysis when cyt c is in its oxidized form; this has also been confirmed by a H18A mutant of cyt c. However, the redox pathway is predominant under reducing conditions in the presence of dithiothreitol over the pH range 6–7.4. Interestingly, we found almost 750-fold enhanced KE catalysis by cyt c compared to aqueous buffer. Overall, in addition to providing mechanistic insights, the data reveal an unprecedented catalytic property of cyt c that could be of high importance in an evolutionary perspective considering its role in delineating the phylogenic tree and also towards generating programmable designer biocatalysts.     

A “Three-in-One” Multifunctional Probe for Bcl-2/Mcl-1 Profiling and Visualizing in Situ

Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously in situ. Herein, we report a multifunctional molecular probe (BnN₃-OPD-Alk) by a “three-in-one” molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN₃-OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both in vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.     

Chemical Modification of Phage-Displayed Helix-Loop-Helix Peptides to Construct Kinase-Focused Libraries

Conformationally constrained peptides hold promise as molecular tools in chemical biology and as a new modality in drug discovery. The construction and screening of a target-focused library could be a promising approach for the generation of de novo ligands or inhibitors against target proteins. Here, we have prepared a protein kinase-focused library by chemically modifying helix-loop-helix (HLH) peptides displayed on phage and subsequently tethered to adenosine. The library was screened against aurora kinase A (AurA). The selected HLH peptide Bip - 3 retained the α-helical structure and bound to AurA with a K_D value of 13.7 μM. Bip - 3 and the adenosine-tethered peptide Bip - 3 - Adc provided IC₅₀ values of 103 μM and 7.7 μM, respectively, suggesting that Bip - 3 - Adc bivalently inhibited AurA. In addition, the selectivity of Bip - 3 - Adc to several protein kinases was tested, and was highest against AurA. These results demonstrate that chemical modification can enable the construction of a kinase-focused library of phage-displayed HLH peptides.     

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.     

Quest for the Crypto-phosphoproteome

Protein phosphorylation is one of the most studied post-translational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically less-stable subset of the phosphosites, which we call the crypto-phosphoproteome, has remained underexplored due to technological challenges. In this Viewpoint, we briefly summarize the current understanding of these elusive protein phosphorylations and identify the missing pieces for future studies.     

Quantification of secondary structure prediction improvement using multiple alignments

The use of multiple sequence alignments for secondary structurepredictions is analysed. Seven different protein families, containingonly sequences of known structure, were considered to providea range of alignment and prediction conditions. Using alignmentsobtained by spatial superposition of main chain atoms in knowntertiary protein structures allowed a mean of 8% in secondarystructure prediction accuracy, when compared to those obtainedfrom the individual sequences. Substitution of these alignmentsby those determined directly from an automated sequence alignmentalgorithm showed variations in the prediction accuracy whichcorrelated with the quality of the multiple alignments and distanceof the primary sequence. Secondary structure predictions canbe reliably improved using alignments from an automatic alignmentprocedure with a mean increase of 6.87percnt;, giving an overallprediction accuracy of 68.5%, if there is a minimum of 25% sequenceidentity between all sequences in a family.     

大豆蛋白/粘胶共混纤维的结构与性能

采用湿法纺丝制备大豆蛋白/粘胶共混纤维,分析测试了共混纤维的结构形态与物理机械性能的关系,并与其他类型的蛋白质改性纤维进行了比较。实验结果表明共混纤维的力学性能与蛋白质比例有关,随着蛋白质质量分数的增加,纤维的强度降低;共混纤维的截面形状和黏胶纤维相似,但表面沟槽更为明显。     

酸、碱溶液对脱脂蚕蛹的解剖结构影响

将脱脂蚕蛹分别置于清水、1%盐酸溶液和1%氢氧化钠溶液中,100℃浸泡处理20 min,采用电子光学显微分析技术观察脱脂蚕蛹的解剖结构。结果表明:①清水浸泡,蛋白质、甲壳素膨胀;②盐酸溶液浸泡,在蛋白质、甲壳素膨胀的同时,有蛋白的部分分解;③氢氧化钠溶液浸泡,蛋白质、甲壳素膨胀,蛋白质溶解,并伴有部分蛋白的分解。碱对蛋白层与甲壳层的层间有劈开作用。碱液能实现蛋白层与甲壳层的剥离。     

Association of seed size with genotypic variation in the chemical constituents of soybeans

Ten soybean genotypes grown in 1992 with seed size ranging from 7.6 to 30.3 g/100 seeds and maturity group V or VI were selected and tested for oil and protein content and for fatty acid composition. In these germplasm, protein varied from 39.5 to 50.2%, oil, 16.3 to 21.6%, and protein plus oil, 59.7 to 67.5%. Percentages of individual fatty acids relative to total fatty acids varied as follows: palmitic, 11.0 to 12.8; stearic, 3.2 to 4.7; oleic, 17.6 to 24.2; linoleic, 51.1 to 56.3 and linolenic, 6.9 to 10.0. Seed size showed no significant correlations with individual saturated fatty acids, protein or oil content. However, significant correlations were found between seed size and individual unsaturated fatty acids: positive with oleic, and negative with linoleic and linolenic. Oil and protein content were negatively correlated with each other. Among the major fatty acids, only the unsaturated were significantly correlated with each other: negative between oleic and linoleic or linolenic, and positive between linoleic and linolenic. A subsequent study with soybeans grown in 1993 generally confirmed these findings. Variation in relative percentages of unsaturated fatty acids andr values for most pairs of relationships were even higher than those obtained from the 1992 crop. Presented at the 85th AOCS Annual Meeting and Expo, Atlanta, Georgia, May 8–12, 1994.