Analysis of a series of phenylalanine 57 mutants of the adipocyte lipid- binding protein

Melanie A. Simpson, David A. Bernlohr

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP ≃ F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP ≃ F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.

Original languageEnglish (US)
Pages (from-to)10980-10986
Number of pages7
JournalBiochemistry
Volume37
Issue number31
DOIs
StatePublished - Aug 4 1998

Fingerprint

Fatty Acid-Binding Proteins
Phenylalanine
Ligands
Apoproteins
Fatty Acids
Denaturation
Chemical stability
Oleic Acid
Escherichia coli
Binding Sites
Lipids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Analysis of a series of phenylalanine 57 mutants of the adipocyte lipid- binding protein. / Simpson, Melanie A.; Bernlohr, David A.

In: Biochemistry, Vol. 37, No. 31, 04.08.1998, p. 10980-10986.

Research output: Contribution to journalArticle

Simpson, Melanie A. ; Bernlohr, David A. / Analysis of a series of phenylalanine 57 mutants of the adipocyte lipid- binding protein. In: Biochemistry. 1998 ; Vol. 37, No. 31. pp. 10980-10986.
@article{329d7adb39614cecaf7d47fb64bc3b6a,
title = "Analysis of a series of phenylalanine 57 mutants of the adipocyte lipid- binding protein",
abstract = "The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP ≃ F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP ≃ F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.",
author = "Simpson, {Melanie A.} and Bernlohr, {David A.}",
year = "1998",
month = "8",
day = "4",
doi = "10.1021/bi980507a",
language = "English (US)",
volume = "37",
pages = "10980--10986",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "31",

}

TY - JOUR

T1 - Analysis of a series of phenylalanine 57 mutants of the adipocyte lipid- binding protein

AU - Simpson, Melanie A.

AU - Bernlohr, David A.

PY - 1998/8/4

Y1 - 1998/8/4

N2 - The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP ≃ F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP ≃ F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.

AB - The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP ≃ F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP ≃ F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.

UR - http://www.scopus.com/inward/record.url?scp=0032483133&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032483133&partnerID=8YFLogxK

U2 - 10.1021/bi980507a

DO - 10.1021/bi980507a

M3 - Article

C2 - 9692991

AN - SCOPUS:0032483133

VL - 37

SP - 10980

EP - 10986

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 31

ER -