5 research outputs found
Dynamics of MutSāMismatched DNA Complexes Are Predictive of Their Repair Phenotypes
MutS
recognizes baseābase mismatches and base insertions/deletions
(IDLs) in newly replicated DNA. Specific interactions between MutS
and these errors trigger a cascade of proteināprotein interactions
that ultimately lead to their repair. The inability to explain why
different DNA errors are repaired with widely varying efficiencies <i>in vivo</i> remains an outstanding example of our limited knowledge
of this process. Here, we present single-molecule FoĢrster resonance
energy transfer measurements of the DNA bending dynamics induced by <i>Thermus aquaticus</i> MutS and the E41A mutant of MutS, which
is known to have error specific deficiencies in signaling repair.
We compared three DNA mismatches/IDLs (T-bulge, GT, and CC) with repair
efficiencies ranging from high to low. We identify three dominant
DNA bending states [slightly bent/unbent (<b>U</b>), intermediately
bent (<b>I</b>), and significantly bent (<b>B</b>)] and
find that the kinetics of interconverting among states varies widely
for different complexes. The increased stability of MutSāmismatch/IDL
complexes is associated with stabilization of <b>U</b> and lowering
of the <b>B</b> to <b>U</b> transition barrier. Destabilization
of <b>U</b> is always accompanied by a destabilization of <b>B</b>, supporting the suggestion that <b>B</b> is a ārequiredā
precursor to <b>U</b>. Comparison of MutS and MutS-E41A dynamics
on GT and the T-bulge suggests that hydrogen bonding to MutS facilitates
the changes in baseābase hydrogen bonding that are required
to achieve the <b>U</b> state, which has been implicated in
repair signaling. Taken together with repair propensities, our data
suggest that the bending kinetics of MutSāmismatched DNA complexes
may control the entry into functional pathways for downstream signaling
of repair
We FRET so You Donāt Have To: New Models of the Lipoprotein Lipase Dimer
Lipoprotein lipase
(LPL) is a dimeric enzyme that is responsible
for clearing triglyceride-rich lipoproteins from the blood. Although
LPL plays a key role in cardiovascular health, an experimentally derived
three-dimensional structure has not been determined. Such a structure
would aid in understanding mutations in LPL that cause familial LPL
deficiency in patients and help in the development of therapeutic
strategies to target LPL. A major obstacle to structural studies of
LPL is that LPL is an unstable protein that is difficult to produce
in the quantities needed for nuclear magnetic resonance or crystallography.
We present updated LPL structural models generated by combining disulfide
mapping, computational modeling, and data derived from single-molecule
FoĢrster resonance energy transfer (smFRET). We pioneer the
technique of smFRET for use with LPL by developing conditions for
imaging active LPL and identifying positions in LPL for the attachment
of fluorophores. Using this approach, we measure LPLāLPL intermolecular
interactions to generate experimental constraints that inform new
computational models of the LPL dimer structure. These models suggest
that LPL may dimerize using an interface that is different from the
dimerization interface suggested by crystal packing contacts seen
in structures of pancreatic lipase
We FRET so You Donāt Have To: New Models of the Lipoprotein Lipase Dimer
Lipoprotein lipase
(LPL) is a dimeric enzyme that is responsible
for clearing triglyceride-rich lipoproteins from the blood. Although
LPL plays a key role in cardiovascular health, an experimentally derived
three-dimensional structure has not been determined. Such a structure
would aid in understanding mutations in LPL that cause familial LPL
deficiency in patients and help in the development of therapeutic
strategies to target LPL. A major obstacle to structural studies of
LPL is that LPL is an unstable protein that is difficult to produce
in the quantities needed for nuclear magnetic resonance or crystallography.
We present updated LPL structural models generated by combining disulfide
mapping, computational modeling, and data derived from single-molecule
FoĢrster resonance energy transfer (smFRET). We pioneer the
technique of smFRET for use with LPL by developing conditions for
imaging active LPL and identifying positions in LPL for the attachment
of fluorophores. Using this approach, we measure LPLāLPL intermolecular
interactions to generate experimental constraints that inform new
computational models of the LPL dimer structure. These models suggest
that LPL may dimerize using an interface that is different from the
dimerization interface suggested by crystal packing contacts seen
in structures of pancreatic lipase
We FRET so You Donāt Have To: New Models of the Lipoprotein Lipase Dimer
Lipoprotein lipase
(LPL) is a dimeric enzyme that is responsible
for clearing triglyceride-rich lipoproteins from the blood. Although
LPL plays a key role in cardiovascular health, an experimentally derived
three-dimensional structure has not been determined. Such a structure
would aid in understanding mutations in LPL that cause familial LPL
deficiency in patients and help in the development of therapeutic
strategies to target LPL. A major obstacle to structural studies of
LPL is that LPL is an unstable protein that is difficult to produce
in the quantities needed for nuclear magnetic resonance or crystallography.
We present updated LPL structural models generated by combining disulfide
mapping, computational modeling, and data derived from single-molecule
FoĢrster resonance energy transfer (smFRET). We pioneer the
technique of smFRET for use with LPL by developing conditions for
imaging active LPL and identifying positions in LPL for the attachment
of fluorophores. Using this approach, we measure LPLāLPL intermolecular
interactions to generate experimental constraints that inform new
computational models of the LPL dimer structure. These models suggest
that LPL may dimerize using an interface that is different from the
dimerization interface suggested by crystal packing contacts seen
in structures of pancreatic lipase
We FRET so You Donāt Have To: New Models of the Lipoprotein Lipase Dimer
Lipoprotein lipase
(LPL) is a dimeric enzyme that is responsible
for clearing triglyceride-rich lipoproteins from the blood. Although
LPL plays a key role in cardiovascular health, an experimentally derived
three-dimensional structure has not been determined. Such a structure
would aid in understanding mutations in LPL that cause familial LPL
deficiency in patients and help in the development of therapeutic
strategies to target LPL. A major obstacle to structural studies of
LPL is that LPL is an unstable protein that is difficult to produce
in the quantities needed for nuclear magnetic resonance or crystallography.
We present updated LPL structural models generated by combining disulfide
mapping, computational modeling, and data derived from single-molecule
FoĢrster resonance energy transfer (smFRET). We pioneer the
technique of smFRET for use with LPL by developing conditions for
imaging active LPL and identifying positions in LPL for the attachment
of fluorophores. Using this approach, we measure LPLāLPL intermolecular
interactions to generate experimental constraints that inform new
computational models of the LPL dimer structure. These models suggest
that LPL may dimerize using an interface that is different from the
dimerization interface suggested by crystal packing contacts seen
in structures of pancreatic lipase