2004
DOI: 10.1073/pnas.0405987101
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New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels

Abstract: To understand why the classical two-state allosteric model of Monod, Wyman, and Changeux explains cooperative oxygen binding by hemoglobin but does not explain changes in oxygen affinity by allosteric inhibitors, we have investigated the kinetic properties of unstable conformations transiently trapped by encapsulation in silica gels. Conformational trapping reveals that after nanosecond photodissociation of carbon monoxide a large fraction of the subunits of the T quaternary structure has kinetic properties al… Show more

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Cited by 108 publications
(158 citation statements)
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“…[5][6][7][8][9][10][11][12][13] In addition, tertiary and quaternary conformational changes in heme proteins, such as myoglobin (Mb) and hemoglobin (Hb), can be inhibited or dramatically slowed, which allows these proteins to be "trapped" and studied in non-equilibrium structural conformations. [14][15][16][17][18][19][20][21][22] The origin of the stability imparted by the silica sol-gel matrix is complex. Since the mean pore diameters in aged wet sol-gels (typically <10 nm) are comparable to the diameters of Mb and Hb, the pore walls could physically restrict protein motions.…”
Section: Introductionmentioning
confidence: 99%
“…[5][6][7][8][9][10][11][12][13] In addition, tertiary and quaternary conformational changes in heme proteins, such as myoglobin (Mb) and hemoglobin (Hb), can be inhibited or dramatically slowed, which allows these proteins to be "trapped" and studied in non-equilibrium structural conformations. [14][15][16][17][18][19][20][21][22] The origin of the stability imparted by the silica sol-gel matrix is complex. Since the mean pore diameters in aged wet sol-gels (typically <10 nm) are comparable to the diameters of Mb and Hb, the pore walls could physically restrict protein motions.…”
Section: Introductionmentioning
confidence: 99%
“…Interest in Hb continues to the present, with new experimental and theoretical methods aimed at characterizing intermediate ligation states of the molecule (3)(4)(5) and at gaining a deeper understanding of its allosteric transition (6)(7)(8)(9)(10)(11).…”
mentioning
confidence: 99%
“…In these experiments, the sample is exposed to the cw laser for various times, suddenly turned off, and the kinetics of CO rebinding to unliganded subunits is measured by optical absorption. Because the gel traps partially liganded T for many minutes, all of the tetramers are in the T quaternary structure during the experiment, as shown previously (34). Fig.…”
Section: Resultsmentioning
confidence: 54%
“…Confirmation of Tertiary r in Liganded Quaternary T. As a control and to confirm our interpretation of r tertiary in T quaternary in the pulsed laser photolysis experiments of Viappiani et al (34) (Fig. 2), we first carried out cw photolysis experiments on the T quaternary structure in the absence of allosteric effectors (T − ) (Fig.…”
Section: Resultsmentioning
confidence: 99%
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