Effect of disruption of Pichia pastoris YPS1 gene on viability and production of recombinant proteins

In the present study, we constructed a Pichia pastoris mutant strain PS111 that lacks one member of the yapsin family through disruption of the YPS1 gene coding for aspartic protease yapsin 1. Under normal growth conditions, the PS111 mutant strain did not show detectable growth defects. Unlike the S. cerevisiae yps1 mutant, the P. pastoris PS111 strain showed no sensitivity when grown in the presence of CaCl₂, elevated temperature (37°C), under acid (pH 4.9) and alkaline (pH 8.3) conditions. Unlike the S. cerevisiae, the P. pastoris yps1 mutant showed decreased growth phenotype induced by cell wall-perturbing reagent sodium dodecyl sulfate (SDS) only when the concentration of SDS was increased by ten times. The use of the yps1 disruptant to produce human interferon alpha16 (hINF-α16) prevents proteolysis, which occurs in the wildtype strain. It was found that the degradation of recombinant protein Alburon composed of human serum albumin (HSA) and hINF-α16 was slightly decreased in the strain lacking yapsin 1.     

Construction of protease-deficient Candida boidinii strains useful for recombinant protein production: cloning and disruption of proteinase A gene (PEP4) and proteinase B gene (PRBI)

The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRBI gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRBI genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4delta and the pep4delta prb1delta strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.     

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43kDa and 66.2kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60h of induction during culture, it was found that the degradation product of around 66.2kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).     

PEP4 Gene Function Is Required for Expression of Several Vacuolar Hydrolases in SACCHAROMYCES CEREVISIAE

The pep4-3 mutation results in a 90–95% reduction in the levels of five vacuolar hydrolases in yeast, including proteinases A and B, carboxypeptidase Y, RNase(s) and the repressible alkaline phosphatase. The mutation is without effect on two secreted glycoproteins, on an enzyme of the vacuolar membrane, and on a proteinase located outside of the vacuole. Mutations at the PEP4 locus exhibit a dosage effect on the levels of some, but not all, of the enzymes whose expression requires the function of the gene.     

KRT8, FAF1 and PTH1R gene polymorphisms are associated with leg weakness traits in pigs

The liability to lesions of dysfunctions of bone and joints in pigs, summarized as leg weakness and mostly expressed as osteochondrosis, is an animal welfare and economic issue in pig production. The objective of this study was to identify polymorphisms in the functional and positional candidate genes keratin 8 (KRT8), Fas-associated factor 1 (FAF1) and parathyroid hormone type I receptor (PTH1R) and to evaluate their association with leg weakness traits. Therefore, osteochondrosis lesions were scored in animals of a Duroc × Pietrain F2 population (DuPi; n = 310) and commercial herds of the breed Large White (n = 298). In addition, bone mineralization traits were observed in DuPi population. SNPs were identified in genes KRT8 (g.8,039G > A), FAF1 (g.380,914T > C) and PTH1R (c.1,672C > T). KRT8 showed significant association with bone mineral density and content (P ≤ 0.05). FAF1 was association with OC lesions score of all joints inspected (P ≤ 0.05). PTH1R showed significant dominance effects on OC lesion scores of the distal femur articular cartilage (P = 0.01) and epiphysis of the distal ulna (P = 0.05) as well as sums of scores of all joints (OCsum, P = 0.04) and assignment to groups of either severely or gently affected animals (OCcat, P = 0.01). This study reveals clear genetic-statistical evidence for a link of KRT8, FAF1 and PTH1R with some of leg weakness related traits in pigs.     

Genetic Properties of Mutations at the PEP4 Locus in SACCHAROMYCES CEREVISIAE

Yeast cells that inherit mutations at the PEP4 locus exhibit a pronounced phenotypic lag in the expression of the mutant phenotype imparted by these mutations. This lag appears to extend to all of the enzymes that are affected by the pep4-3 mutation. For at least two of the enzymatic activities, phenotypic lag shows mitotic cosegregation. Phenotypic lag is found for meiotic progeny and for mitotic segregants from heterokaryons. The phenotypic lag in the expression of the carboxypeptidase Y deficiency is abolished by nonsense mutations in either PRC1, the structural gene for carboxypeptidase Y, or PRB1, the structural gene for proteinase B. Models to explain these observations are proposed.     

Candida albicans PEP12 Is Required for Biofilm Integrity and In Vivo Virulence

To investigate the role of the prevacuolar secretion pathway in biofilm formation and virulence in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae prevacuolar trafficking gene PEP12. C. albicans PEP12 encodes a deduced t-SNARE that is 28% identical to S. cerevisiae Pep12p, and plasmids bearing C. albicans PEP12 complemented the abnormal vacuolar morphology and temperature-sensitive growth of an S. cerevisiae pep12 null mutant. The C. albicans pep12 Δ null mutant was defective in endocytosis and vacuolar acidification and accumulated 40- to 60-nm cytoplasmic vesicles near the plasma membrane. Secretory defects included increased extracellular proteolytic activity and absent lipolytic activity. The pep12Δ null mutant was more sensitive to cell wall stresses and antifungal agents than the isogenic complemented strain or the control strain DAY185. Notably, the biofilm formed by the pep12Δ mutant was reduced in overall mass and fragmented completely upon the slightest disturbance. The pep12Δ mutant was markedly reduced in virulence in an in vitro macrophage infection model and an in vivo mouse model of disseminated candidiasis. These results suggest that C. albicans PEP12 plays a key role in biofilm integrity and in vivo virulence.In Saccharomyces cerevisiae, distinct secreted marker proteins are trafficked differentially through a prevacuolar compartment (PVC) prior to exocytosis (14). Furthermore, prevacuolar protein sorting genes play an important role in cargo transport in the prevacuolar branch of the exocytic pathway in S. cerevisiae (13, 15). By isolating dense- and light-vesicle populations in S. cerevisiae vps1 sec6-4, vps4 sec6-4, and pep12 sec6-4 mutants, it was observed that mutants blocked in this prevacuolar pathway missort marker proteins that are normally found in high-density post-Golgi compartment vesicles into low-density vesicles (15). Gurunathan et al. (13) also demonstrated these findings for vps1 and pep12 mutants with a late secretory mutant (snc1) background similar to that of the sec6-4 strains. These results indicate that some exocytic cargo, including the conditionally regulated soluble secretory proteins invertase and acid phosphatase, are differentially sorted through a PVC prior to exocytosis in the model yeast S. cerevisiae.To study the prevacuolar branch of exocytosis in Candida albicans and its role in virulence, we have previously cloned and analyzed the C. albicans prevacuolar trafficking genes VPS1 and VPS4. We demonstrated that C. albicans VPS4 is required for extracellular secretion of Sap2p and Sap4-6p and for virulence in an in vivo model of disseminated candidiasis (19, 20). C. albicans VPS1 is required for Sap2p secretion and biofilm formation (4). Interestingly, although the C. albicans null mutant lacking VPS4 forms a biofilm that is denser than that formed by the isogenic reintegrant strain, the conditional mutant lacking VPS1 expression forms a patchy biofilm of reduced density (4, 34). Thus, it appears that interference with normal prevacuolar trafficking affects both the secretion of virulence-associated proteins and biofilm formation.S. cerevisiae PEP12 encodes a 288-amino-acid syntaxin which regulates docking of Golgi compartment-derived transport vesicles at the PVC (3). Pep12p interacts with the v-SNARE Vti1p, and overexpression of Pep12p suppresses extracellular missorting of carboxypeptidase in the vti1 mutant (37). The S. cerevisiae pep12 null mutant displays a temperature-sensitive growth defect and is characterized by an enlarged vacuole with morphology defined as class D (3). A search of the C. albicans genome database identified a structural homolog of S. cerevisiae PEP12. Thus, the experiments described below were designed to determine whether the C. albicans PEP12 homolog is functionally homologous to S. cerevisiae PEP12 and to investigate its role in secretion, biofilm formation, and virulence.     

Proteolytic inactivation of the NADP-dependent glutamate dehydrogenase in proteinase-deficient mutants of Saccharomyces cerevisiae

The proteolytic inactivation of NADP-dependent glutamate dehydrogenase (l-glutamate: NADP⁺ oxidoreductase, EC 1.4.1.4) during carbon starvation was studied using several proteinase-deficient mutants of Saccharomyces cerevisiae. Strains bearing mutations in the structural genes for proteinase B, proteinase C (carboxypeptidase Y), or in both genes catalyzed the inactivation and initial proteolytic cleavage of NADP-glutamate dehydrogenase at a rate indistinguishable from that of the wild-type parent strain. In addition, the pleiotropic mutation, pep4-3, which results in a deficiency for proteinases A, B, and C, did not affect the inactivation or initial proteolytic cleavage of NADP-glutamate dehydrogenase.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.     

Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.     

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Enhancement of free fatty acid production in Saccharomyces cerevisiae by control of fatty acyl-CoA metabolism

Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

A new, vitamin D-based, multidimensional nomogram for the diagnosis of primary hyperparathyroidism

ObjectiveTo refine the diagnostic criteria for primary hyperparathyroidism (1°HPT) to identify atypical patients, in whom serum calcium, parathyroid hormone (PTH), or both are within the “normal” range.MethodsTotal serum calcium, intact PTH, and 25-hydroxyvitamin D [25(OH)D] levels were measured in patients with 1°HPT and healthy patient groups. Multivariate analysis of healthy patient data first identified factors that significantly affected PTH levels and defined a new PTH reference range with a mathematical model. That nomogram was then validated for prediction of atypical 1°HPT in patients with surgically confirmed disease.ResultsOn multivariate analysis, calcium (P = .0002), 25(OH)D (P < .0001), and age (P = .015) independently affected PTH. With these variables, we created a 4-dimensional nomogram that distinguished normal patients from those with hyperparathyroid states. Mathematically, this nomogram predicts 1°HPT when the measured serum PTH value is higher than PTH calculated by the following formula: PTH (pg/mL) = 120-[6 × calcium (mg/dL)]-[0.52 × 25(OH)D (ng/mL)] + [0.26 × patient age (years)]. When applied to our surgical group of patients, this nomogram successfully identified 100% of patients (238 of 238) with classic 1°HPT, 84% (64 of 76) with normocalcemic 1°HPT, and 54% (20 of 37) with 1°HPT and normal PTH.ConclusionThis study uniquely defines a patientspecific upper limit of normal for PTH based on the readily available variables of serum calcium, 25(OH)D, and patient age. Our nomogram may allow for more rapid definitive diagnosis and treatment of 1°HPT in patients with atypical presentations. (Endocr Pract. 2012;18:124-131)     

Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein

Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be K_piperine?=?5.7 ± .2 × 10⁵ M^?1 and K_piperine = 9.3± .25 × 10⁴ M^?1 which correspond to the free energy of ?7.8 and ?6.71 kcal M^?1at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA–piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA–piperine complex reaches equilibration state at around 3 ns, which prove that the HSA–piperine complex is stable in nature.     

Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein

Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be K_Umb-1=3.1 ± .01 × 10⁴ M^?1, K_Umb-2 = 7 ± .01 × 10⁴ M^?1, which corresponds to ?6.1 and ?6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of K_Umb-1-AGP=3.1 ± .01 × 10³ M^?1 and K_Umb-2-AGP = 4.6 ± .01 × 10³ M^?1. Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster’s theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1–HSA and Umb-2–HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.