Kane Bryant J, Murphy Kyle V, Sode Koji
Lampe Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North Carolina State University, Chapel Hill, North Carolina, USA.
Protein Sci. 2025 May;34(5):e70129. doi: 10.1002/pro.70129.
β-hydroxybutyrate binding proteins (BHBBPs) are a newly identified group of periplasmic solute-binding proteins (SBPs) that interact with β-hydroxybutyrate (BHB), a key physiological metabolite. In this study, we systematically characterized the interaction properties of both previously reported and newly identified BHBBPs, including "NovoS" and "EDC10" from Gram-negative bacteria. Following recombinant production, we assessed the specificity and affinity of these proteins against a library of 23 different metabolites using a label-free derivative of differential scanning fluorimetry (nanoDSF). Positive interactions were further evaluated for their binding affinity via tryptophan fluorescence spectroscopy, which confirmed D/L-BHB as the preferred ligand for all proteins, with slight enantioselectivity. BHBBPs also exhibited binding to other compounds such as acetoacetate, D/L-α-hydroxybutyrate, L-lactate, and pyruvate, albeit with reduced affinity. These findings expand the classification of BHBBPs, suggesting that similar proteins and associated transporters may be widespread in prokaryotes involved in the carbon cycle of polyhydroxybutyrate. Guided by the crystal structure of the homologous BMA2936 protein, we introduced targeted point mutations in conserved polar residues of the BHBBPs EDC24 and NovoS. It was determined through this experimental pipeline that their affinity towards BHB was reduced by a factor between 25 and 750, shifting their binding constants towards the millimolar range. Collectively, the affinities of both wild-type and mutant proteins span 4 orders of magnitude, from nanomolar to millimolar recognition of BHB. Leveraging the versatility of SBP-based biosensing, these receptors and their wide affinity range could facilitate the development of effective bioanalytical tools for BHB detection in diverse physiological environments.
β-羟基丁酸结合蛋白（BHBBPs）是一组新发现的周质溶质结合蛋白（SBPs），它们与关键生理代谢物β-羟基丁酸（BHB）相互作用。在本研究中，我们系统地表征了先前报道的和新鉴定的BHBBPs的相互作用特性，包括来自革兰氏阴性菌的“NovoS”和“EDC10”。重组表达后，我们使用差示扫描荧光法的无标记衍生物（nanoDSF）评估了这些蛋白对23种不同代谢物文库的特异性和亲和力。通过色氨酸荧光光谱进一步评估阳性相互作用的结合亲和力，这证实了D/L-BHB是所有蛋白的首选配体，具有轻微的对映选择性。BHBBPs也表现出与其他化合物如乙酰乙酸、D/L-α-羟基丁酸、L-乳酸和丙酮酸的结合，尽管亲和力较低。这些发现扩展了BHBBPs的分类，表明类似的蛋白和相关转运体可能广泛存在于参与聚羟基丁酸碳循环的原核生物中。以同源BMA2936蛋白的晶体结构为指导，我们在BHBBPs EDC24和NovoS的保守极性残基中引入了靶向点突变。通过该实验流程确定，它们对BHB的亲和力降低了25至750倍，使其结合常数向毫摩尔范围移动。总体而言，野生型和突变型蛋白的亲和力跨度为4个数量级，从对BHB的纳摩尔识别到毫摩尔识别。利用基于SBP的生物传感的多功能性，这些受体及其广泛的亲和力范围可以促进开发用于在不同生理环境中检测BHB的有效生物分析工具。
