Cytochrome c recognition of immobilized, orientational variants of cytochrome b5: Direct force and equilibrium binding measurements

C. Yeung, T. Purves, A. A. Kloss, T. L. Kuhl, Stephen Sligar, Deborah E Leckband

Research output: Contribution to journalArticle

Abstract

Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.

Original languageEnglish (US)
Pages (from-to)6829-6836
Number of pages8
JournalLangmuir
Volume15
Issue number20
DOIs
StatePublished - Sep 28 1999

Fingerprint

Cytochromes b5
cytochromes
Cytochromes c
Force measurement
Proteins
Electrostatics
Membrane Proteins
proteins
Electrostatic force
electrostatics
Surface plasmon resonance
Monolayers
Spectroscopy
interactions
Electrons
surface plasmon resonance
manipulators
electron transfer
Surface potential

ASJC Scopus subject areas

  • Materials Science(all)
  • Condensed Matter Physics
  • Surfaces and Interfaces
  • Spectroscopy
  • Electrochemistry

Cite this

Cytochrome c recognition of immobilized, orientational variants of cytochrome b5 : Direct force and equilibrium binding measurements. / Yeung, C.; Purves, T.; Kloss, A. A.; Kuhl, T. L.; Sligar, Stephen; Leckband, Deborah E.

In: Langmuir, Vol. 15, No. 20, 28.09.1999, p. 6829-6836.

Research output: Contribution to journalArticle

@article{e27dd923ca1d4149aeb015604ab7133d,
title = "Cytochrome c recognition of immobilized, orientational variants of cytochrome b5: Direct force and equilibrium binding measurements",
abstract = "Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.",
author = "C. Yeung and T. Purves and Kloss, {A. A.} and Kuhl, {T. L.} and Stephen Sligar and Leckband, {Deborah E}",
year = "1999",
month = "9",
day = "28",
doi = "10.1021/la990019j",
language = "English (US)",
volume = "15",
pages = "6829--6836",
journal = "Langmuir",
issn = "0743-7463",
publisher = "American Chemical Society",
number = "20",

}

TY - JOUR

T1 - Cytochrome c recognition of immobilized, orientational variants of cytochrome b5

T2 - Direct force and equilibrium binding measurements

AU - Yeung, C.

AU - Purves, T.

AU - Kloss, A. A.

AU - Kuhl, T. L.

AU - Sligar, Stephen

AU - Leckband, Deborah E

PY - 1999/9/28

Y1 - 1999/9/28

N2 - Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.

AB - Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.

UR - http://www.scopus.com/inward/record.url?scp=0032683345&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032683345&partnerID=8YFLogxK

U2 - 10.1021/la990019j

DO - 10.1021/la990019j

M3 - Article

AN - SCOPUS:0032683345

VL - 15

SP - 6829

EP - 6836

JO - Langmuir

JF - Langmuir

SN - 0743-7463

IS - 20

ER -