Efficient identification of photolabelled amino acid residues by combining immunoaffinity purification with MS: revealing the semotiadil-binding site and its relevance to binding sites for myristates in domain III of human serum albumin

To identify photoaffinity-labelled amino acid residue(s), we devised an effective method utilizing immunoaffinity purification of photolabelled fragments, followed by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) MS and nanoelectrospray ionization tandem MS (nano-ESI-MS/MS) analysis. Human serum albumin (HSA) was photolabelled with an azidophenyl derivative of semotiadil, FNAK {(+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-(3-azidophenoxy)-ethyl]amino]propoxyl]phenyl]-4-methyl-2H-1,4-benzothiazin-3-(4H)-one}, since HSA is a major binding protein for semotiadil in serum. After lysyl endopeptidase digestion, photolabelled HSA fragments were adsorbed selectively on to Sepharose beads on which an anti-semotiadil antibody was immobilized, and fractions were eluted quantitatively by 50% acetonitrile/10mM HCl. MALDI-TOF MS analysis of the eluted fraction showed that it contained two photolabelled fragments of m/z 2557.54 (major) and 1322.44 (minor), corresponding to Lys-414—Lys-432 and Ala-539—Lys-545, respectively. Further nano-ESI-MS/MS analysis revealed that Lys-414 was the photolabelled amino acid residue in fragment 414–432 and Lys-541 was a likely candidate in fragment 539–545. Based on the photolabelling results, we constructed a three-dimensional model of the FNAK—HSA complex, revealing that FNAK resides in a pocket that overlaps considerably with myristate (Myr)-binding sites, Myr-3 and −4, by comparison with crystallographic data of HSA—Myr complexes described in Curry, Mandelkow, Brick and Franks (1998) Nat. Struct. Biol. 5, 827–835. Moreover, addition of Myr increased photo-incorporation into Lys-414, whereas incorporation into Lys-541 decreased under conditions of [Myr]/[HSA]<1. Further addition of Myr, however, uniformly decreased photo-incorporation into both Lys residues. These results indicate that FNAK labelling can also be used to monitor Myr binding in domain III. An interpretation for the concomitant local conformational change of HSA is provided.

Download Full-text

Influence of mutations caused by radiation exposure on the bilirubin binding sites of human serum albumin

Proceedings of the National Academy of Sciences of Belarus Medical series ◽

10.29235/1814-6023-2021-18-1-46-57 ◽

2021 ◽

Vol 18 (1) ◽

pp. 46-57

Author(s):

V. V. Poboinev ◽

V. V. Khrustalev ◽

A. N. Stojarov ◽

T. A. Khrustaleva

Keyword(s):

Amino Acid ◽

Secondary Structure ◽

Human Serum Albumin ◽

Serum Albumin ◽

Radiation Exposure ◽

Human Serum ◽

Binding Sites ◽

Amino Acid Substitutions ◽

Amino Acid Residues ◽

Bilirubin Binding

In this article we analyze the bilirubin binding sites of human serum albumin from the point of view of the secondary structure instability, as well as the effect of amino acid substitutions caused by radiation exposure on the ability of albumin to bind bilirubin-IX-alpha. Based on calculations of binding energy and inhibition constants of bilirubin-albumin complexes before and after the amino acid substitutions, it was found that amino acid substitutions have different effects on the ability of human serum albumin to bind bilirubin. Amino acid substitutions Asp269-Gly269 (Nagasaki-1), Glu354-Lys354 (Hiroshima-1), Asp375-Asn375 (Nagasaki-2) reduce the binding free energy of bilirubin with human serum albumin, and the amino acid substitutions His3-Gln3 (Nagasaki-3) and Glu382-Lys382 (Hiroshima-2) increase it during molecular docking with the corresponding areas of the protein surface. The inhibition constants are significantly higher than with known binding sites. In general, mutations caused by radiation exposure cannot effect on bilirubin binding sites of human serum albumin, since the amino acid residues that are replaced do not interact with the amino acid residues from the binding sites (Leu115, Arg117, Phe134, Tyr138, Ile142, Phe149, Phe157, Tyr161, Arg186, Lys190, Lys240, Arg222). All amino acid residues from known binding sites are located in stable elements of the secondary structure of human serum albumin.The data obtained are important for understanding the impact of radiation exposure on the development of bilirubin encephalopathy in the population of the Chernobyl region and Japan.

Download Full-text

The fatty-acid-induced conformational states of human serum albumin investigated by means of multiple co-binding of protons and oleic acid

Biochemical Journal ◽

10.1042/bj2500443 ◽

1988 ◽

Vol 250 (2) ◽

pp. 443-446 ◽

Cited By ~ 11

Author(s):

J H M Dröge ◽

L H M Janssen ◽

J Wilting

Keyword(s):

Oleic Acid ◽

Fatty Acid ◽

Amino Acid ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Binding Sites ◽

Conformational Transitions ◽

Amino Acid Residues ◽

Ph Stat

The binding of oleic acid to human serum albumin causes progressive changes in (a) the pK of some amino acid residues, as detected by pH-stat titration and (b) the induced molar ellipticities of albumin-bound drugs (diazepam and oxyphenbutazone), as measured by c.d. It is concluded that albumin undergoes several conformational transitions as the amount of oleic acid bound increases from 0 to about 9 molecules/molecule of protein. At least three different conformations of the protein seem to be involved. These conformations can be linked with the three classes of oleic acid-binding sites on albumin.

Download Full-text

Oxidative Alteration of Trp-214 and Lys-199 in Human Serum Albumin Increases Binding Affinity with Phenylbutazone: A Combined Experimental and Computational Investigation

International Journal of Molecular Sciences ◽

10.3390/ijms19102868 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2868 ◽

Cited By ~ 1

Author(s):

Luiza Bertozo ◽

Ernesto Tavares Neto ◽

Leandro Oliveira ◽

Valdecir Ximenes

Keyword(s):

Amino Acid ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Interaction Energy ◽

Score Function ◽

Oxidation Products ◽

Amino Acid Residues ◽

Computational Investigation ◽

A Current

Human serum albumin (HSA) is a target for reactive oxygen species (ROS), and alterations of its physiological functions caused by oxidation is a current issue. In this work, the amino-acid residues Trp-214 and Lys-199, which are located at site I of HSA, were experimentally and computationally oxidized, and the effect on the binding constant with phenylbutazone was measured. HSA was submitted to two mild oxidizing reagents, taurine monochloramine (Tau-NHCl) and taurine dibromamine (Tau-NBr2). The oxidation of Trp-214 provoked spectroscopic alterations in the protein which were consistent with the formation of N′-formylkynurenine. It was found that the oxidation of HSA by Tau-NBr2, but not by Tau-NHCl, provoked a significant increase in the association constant with phenylbutazone. The alterations of Trp-214 and Lys-199 were modeled and simulated by changing these residues using the putative oxidation products. Based on the Amber score function, the interaction energy was measured, and it showed that, while native HSA presented an interaction energy of −21.3 kJ/mol, HSA with Trp-214 altered to N′-formylkynurenine resulted in an energy of −28.4 kJ/mol, and HSA with Lys-199 altered to its carbonylated form resulted in an energy of −33.9 kJ/mol. In summary, these experimental and theoretical findings show that oxidative alterations of amino-acid residues at site I of HSA affect its binding efficacy.

Download Full-text

Indomethacin Increases Quercetin Affinity for Human Serum Albumin: A Combined Experimental and Computational Study and Its Broader Implications

International Journal of Molecular Sciences ◽

10.3390/ijms21165740 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5740

Author(s):

Hrvoje Rimac ◽

Tana Tandarić ◽

Robert Vianello ◽

Mirza Bojić

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Binding Site ◽

Binding Sites ◽

Quantum Chemical ◽

Computational Study ◽

The Body ◽

Active Concentration ◽

Insight Into

Human serum albumin (HSA) is the most abundant carrier protein in the human body. Competition for the same binding site between different ligands can lead to an increased active concentration or a faster elimination of one or both ligands. Indomethacin and quercetin both bind to the binding site located in the IIA subdomain. To determine the nature of the HSA-indomethacin-quercetin interactions, spectrofluorometric, docking, molecular dynamics studies, and quantum chemical calculations were performed. The results show that the indomethacin and quercetin binding sites do not overlap. Moreover, the presence of quercetin does not influence the binding constant and position of indomethacin in the pocket. However, binding of quercetin is much more favorable in the presence of indomethacin, with its position and interactions with HSA significantly changed. These results provide a new insight into drug-drug interactions, which can be important in situations when displacement from HSA or other proteins is undesirable or even desirable. This principle could also be used to deliberately prolong or shorten the xenobiotics’ half-life in the body, depending on the desired outcomes.

Download Full-text

The binding affinity of amino acid–protein: hydroxyproline binding site I on human serum albumin

Organic & Biomolecular Chemistry ◽

10.1039/c2ob25967b ◽

2012 ◽

Vol 10 (41) ◽

pp. 8314 ◽

Cited By ~ 7

Author(s):

Ximin Zhou ◽

Wenjuan Lü ◽

Li Su ◽

Yalei Dong ◽

Qianfeng Li ◽

...

Keyword(s):

Amino Acid ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Binding Site ◽

Binding Affinity ◽

Acid Protein ◽

Amino Acid Protein

Download Full-text

Amino acid sequence adjoining the lone tryptophan of human serum albumin. A binding site of the protein

Biochemistry ◽

10.1021/bi00815a003 ◽

1970 ◽

Vol 9 (13) ◽

pp. 2570-2574 ◽

Cited By ~ 77

Author(s):

John B. Swaney ◽

Irving M. Klotz

Keyword(s):

Amino Acid ◽

Human Serum Albumin ◽

Amino Acid Sequence ◽

Serum Albumin ◽

Human Serum ◽

Binding Site

Download Full-text

Dansylation of human serum albumin in the study of the primary binding sites of bilirubin and l-tryptophan

Biochemical Journal ◽

10.1042/bj1810251 ◽

1979 ◽

Vol 181 (1) ◽

pp. 251-253 ◽

Cited By ~ 21

Author(s):

C Jacobsen ◽

J Jacobsen

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Binding Site ◽

Binding Sites ◽

Lysine Residue ◽

Bilirubin Binding ◽

Albumin Molecule ◽

Common Cavity

Binding of bilirubin and of L-tryptophan to dansylated albumins was investigated. Dansylation of less than one lysine residue per molecule of albumin did not affect the bilirubin binding, but decreased the L-tryptophan binding, indicating that dansylation had taken place in or near the l-tryptophan-binding site. Native albumin and albumin-bilirubin 1:1 complex showed the same affinity for L-tryptophan. The results indicate that, although L-tryptophan and bilirubin are bound in the same region, perhaps in a common cavity of the albumin molecule, such a cavity is sufficiently large to contain both ligands.

Download Full-text