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sample_so.med
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UI - 98301406
AU - Ghaemmaghami S
AU - Word JM
AU - Burton RE
AU - Richardson JS
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710, USA.
TI - Folding kinetics of a fluorescent variant of monomeric lambda repressor.
SO - Biochemistry 1998 Jun 23;37(25):9179-85
AB - A tryptophan-containing variant of monomeric lambda repressor has been
made, and its folding kinetics were analyzed at 20 degreesC using
fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation
curves obtained by circular dichroism, fluorescence, and NMR are
superimposable. Stopped-flow analysis indicates that in the absence of
denaturants the folding reaction is complete within the dead-time of the
experiment. Within higher denaturant conditions, where the folding rate
is slower, NMR and stopped-flow agree on the folding and unfolding rates
of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant
folds within 2 ms. Extrapolation indicates that the folding time is 20
micro(s) in the absence of denaturants. All folding and unfolding
reactions displayed monoexponential kinetics, and no burst-phases were
observed. In addition, the thermodynamic parameters Delta G and meq
obtained from the kinetic analysis are consistent with the equilibrium
experiments. The results support a two-state Dleft and right arrow N
folding model.
UI - 98217287
AU - Burton RE
AU - Myers JK
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710, USA.
TI - Protein folding dynamics: quantitative comparison between theory and
experiment.
SO - Biochemistry 1998 Apr 21;37(16):5337-43
AB - The development of a quantitative kinetic scheme is a central goal in
mechanistic studies of biological phenomena. For fast-folding proteins,
which lack experimentally observable kinetic intermediates, a
quantitative kinetic scheme describing the order and rate of events
during folding has yet to be developed. In the present study, the folding
mechanism of monomeric lambda repressor is described using the
diffusion-collision model and estimates of intrinsic alpha-helix
propensities. The model accurately predicts the folding rates of the
wild-type protein and five of eight previously studied Ala left and right
arrow Gly variants and suggests that the folding mechanism is distributed
among multiple pathways that are highly sensitive to the amino acid
sequence. For example, the model predicts that the wild-type protein
folds through a small number of pathways with a folding time of 260
micros. However, the folding of a variant (G46A/G48A) is predicted to
fold through a large number of pathways with a folding time of 12 micros.
Both folding times quantitatively agree with the experimental values at
37 degrees C extrapolated to 0 M denaturant. The quantitative nature of
the diffusion-collision model allows for rigorous experimental tests of
the theory.
UI - 97249294
AU - Burton RE
AU - Huang GS
AU - Daugherty MA
AU - Calderone TL
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710, USA.
TI - The energy landscape of a fast-folding protein mapped by Ala-->Gly
substitutions.
SO - Nature Structural Biology 1997 Apr;4(4):305-10
AB - A moderately stable protein with typical folding kinetics unfolds and
refolds many times during its cellular lifetime. In monomeric lambda
repressor this process is extremely rapid, with an average folded state
lifetime of only 30 milliseconds. A thermostable variant of this protein
(G46A/G48A) unfolds with the wild-type rate, but it folds in
approximately 20 microseconds making it the fastest-folding protein yet
observed. The effects of alanine to glycine substitutions on the folding
and unfolding rate constants of the G46A/G48A variant, measured by
dynamic NMR spectroscopy, indicate that the transition state is an
ensemble comprised of a disperse range of conformations. This structural
diversity in the transition state is consistent with the idea that
folding chains are directed towards the native state by a smooth
funnel-like conformational energy landscape. The kinetic data for the
folding of monomeric lambda repressor can be understood by merging the
new energy landscape view of folding with traditional models. This hybrid
model incorporates the conformational diversity of denatured and
transition state ensembles, a transition state activation energy, and the
importance of intrinsic helical stabilities.
UI - 97336212
AU - Beeser SA
AU - Goldenberg DP
AU - Oas TG
IN - Department of Biology, University of Utah, Salt Lake City 84112, USA.
TI - Enhanced protein flexibility caused by a destabilizing amino acid
replacement in BPTI.
SO - Journal of Molecular Biology 1997 May 30;269(1):154-64
AB - A genetically engineered variant of bovine pancreatic trypsin inhibitor
(Y35G BPTI) has been shown previously by X-ray crystallography to have a
three-dimensional structure dramatically different from that of the
wild-type protein, particularly in the protease-binding region of the
molecule. Yet, the Y35G variant is a potent trypsin inhibitor. Described
here are 15N NMR relaxation studies to compare the backbone dynamics of
Y35G BPTI to those of the wild-type protein. The Tyr35 --> Gly
substitution increased the transverse relaxation rates of more than one
third of all backbone amide groups, but had little effect on the
longitudinal relaxation rates, indicating that the substitution
facilitates relatively slow backbone motions, estimated to be on the
microsecond time-scale. The results indicate that the residues making up
the trypsin-binding site undergo large and relatively slow conformational
changes in solution, estimated to be on the 5 to 20 micros time-scale. It
is thus likely that the crystal structure represents only one of multiple
interconverting conformations in solution, only a fraction of which may
be competent for binding trypsin. The large thermodynamic destabilization
associated with this substitution may arise, in part, from a loss in
cooperativity among the multiple stabilizing interactions that are
normally favored by the highly ordered structure of the wild-type
protein. These results suggest that fully understanding the effects of
amino acid replacements on the functional and thermodynamic properties of
proteins may often require analysis of the dynamic, as well as the
structural, properties of altered proteins.
UI - 97070383
AU - Burton RE
AU - Huang GS
AU - Daugherty MA
AU - Fullbright PW
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, NC
27710, USA.
TI - Microsecond protein folding through a compact transition state.
SO - Journal of Molecular Biology 1996 Oct 25;263(2):311-22
AB - Dynamic NMR methods have been employed to measure the folding and
unfolding rate constants of two extremely fast-folding proteins. lambda
6-85, a truncated, monomeric form of the N-terminal domain of lambda
repressor, refolds with a lifetime of approximately 250 microseconds.
These methods have also been applied to a thermostable lambda 6-85
variant with alanine substituted for glycine residues 46 and 48 in the
third helix (G46A/G48A). Both proteins exhibit linear ln (kf,u) versus
[urea] plots, consistent with two-state folding for both proteins. When
extrapolated to 0M urea, the data indicate that G46A/G48A folds with a
lifetime of less than 20 microseconds. The slopes of the ln (kf,u) versus
[urea] curves (mu and mf) indicate that the modest Gly-->Ala double
mutation dramatically changes the transition state solvent accessibility.
The transition state for lambda 6-85 has a fractional accessibility
(mu/(mu-mf)) of 0.61, whereas the transition state for G46A/G48A is much
more native-like, with a fractional accessibility of 0.16. The
extraordinary change in the folding pathway that these mutations induce
suggests that the intrinsic stability of helix 3 is an important
determinant of the folding mechanism.
UI - 97049122
AU - Calderone TL
AU - Stevens RD
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, NC
27710, USA.
TI - High-level misincorporation of lysine for arginine at AGA codons in a
fusion protein expressed in Escherichia coli.
SO - Journal of Molecular Biology 1996 Oct 4;262(4):407-12
AB - The expression of eukaryotic genes in Escherichia coli is one of the most
frequently used tools of modern science. The arginine codon AGA is a
common codon in eukaryotic genes but is particularly rare in E. coli. We
report here 36 to 42% misincorporation of lysine at three AGA codons in a
well-expressed protein. This misincorporation yields a protein whose
electrospray mass spectrum (ESMS) shows peaks at the expected mass (M),
M-28, M-56 and M-84 with intensities representing 34.5(+/-0.7),
37.5(+/-1.1), 21.2(+/-1.7) and 6.6(+/-0.5) % of the total intensity,
respectively. Replacement of either all three AGA codons or the two
closest to the 3' end of the gene by the more common CGC arginine codon
gave a protein with a single ESMS peak. Misincorporation could also be
eliminated by the co-expression of the tRNA(UCL)Arg gene, argU. These
studies demonstrate that misincorporation of amino acids at rare codons
of recombinant proteins can be far higher than previously thought.
UI - 96224816
AU - Huang GS
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710, USA.
TI - Heat and cold denatured states of monomeric lambda repressor are
thermodynamically and conformationally equivalent.
SO - Biochemistry 1996 May 21;35(20):6173-80
AB - Although the denaturation of proteins by low temperatures is a
well-documented phenomenon, little is known about the molecular details
of the process. In this study, the parameters describing the denaturation
thermodynamics of residues 6-85 of the N-terminal domain of lambda
repressor have been determined by fitting the three-dimensional
thermal-urea denaturation surface obtained by circular dichroism. The
shape of the surface shows cold denaturation at low temperatures and urea
concentrations above 2 M, which allows accurate determination of the
apparent heat capacity of denaturation (delta Cp). Denaturation curves
based on aromatic 1H NMR spectra give identical denaturation curves,
confirming purely twostate folding under all conditions studies. The
denaturation surface can be fit with constant delta Cp and delta In
KD/delta[urea] (KD is the equilibrium constant for denaturation),
consistent with a thermodynamically invariant denatured state. In
addition, the aromatic 1H NMR spectrum of the cold denatured state at 0
degree C in 3 M uea is essentially identical to the spectrum at 70 degree
C in 3 M urea. These observations indicate that the structures of the
cold and heat denatured states, in the presence of 3 M urea, are
thermodynamically and conformationally equivalent.
UI - 95350173
AU - Huang GS
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, NC
27710, USA.
TI - Submillisecond folding of monomeric lambda repressor.
SO - Proceedings of the National Academy of Sciences of the United States of
America 1995 Jul 18;92(15):6878-82
AB - The folding kinetics of a truncated form of the N-terminal domain of
phage lambda repressor [lambda 6-85] has been investigated by using the
technique of dynamic NMR. lambda 6-85 has been shown previously to fold
in a purely two-state fashion. This allows the determination of folding
and unfolding rates from simulation of the exchange-broadened aromatic
resonances of Tyr-22. The folding kinetics were determined over a range
of 1.35 to 3.14 M urea. The urea dependence of both folding and unfolding
rate constants is exponential, suggesting that the rate-determining step
is invariant at the urea concentrations studied. The folding and
unfolding rates extrapolated to 0 M urea at 37 degrees C are 3600 +/- 400
s-1 and 27 +/- 6 s-1, respectively. The observed lambda 6-85 folding rate
constant exceeds that of other fast-folding globular proteins by a factor
of 14-54. The urea dependence of the folding and unfolding rate constants
suggests that the transition state of the rate-determining step is
considerably more exposed to solvent than previously studied
protein-folding transition states. The surprising rapidity of lambda 6-85
folding and unfolding may be the consequence of its all-helical secondary
structure. These kinetic results clearly demonstrate that all of the
fundamental events of protein folding can occur on the submillisecond
time scale.
UI - 95210251
AU - Huang GS
AU - Oas TG
IN - Department of Biochemistry, Duke University Medical Center, Durham, North
Carolina 27710.
TI - Structure and stability of monomeric lambda repressor: NMR evidence for
two-state folding.
SO - Biochemistry 1995 Mar 28;34(12):3884-92
AB - The absence of equilibrium intermediates in protein folding reactions
(i.e., two-state folding) simplifies thermodynamic and kinetic analyses
but is difficult to prove rigorously. We demonstrate a sensitive method
for detecting partially folded species based on using proton chemical
shifts as local probes of structure. The coincidence of denaturation
curves for probes throughout the molecule is a particularly stringent
test for two-state folding. In this study we investigate a new form of
the N-terminal domain of bacteriophage lambda repressor consisting of
residues 6-85 (lambda 6-85) using nuclear magnetic resonance (NMR) and
circular dichroism (CD). This truncated version lacks the residues
required for dimerization and is monomeric under the conditions used for
NMR. Heteronuclear NMR was used to assign the 1H, 15N, and backbone 13C
resonances. The secondary and tertiary structure of lambda 6-85 is very
similar to that reported for the crystal structure of the DNA-bound 1-92
fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mol. Biol. 227,
177-196], as judged by analysis of chemical shifts, amide hydrogen
exchange, amide-alpha coupling constants, and nuclear Overhauser
enhancements. Thermal and urea denaturation studies were conducted using
the chemical shifts of the four aromatic side chains as local probes and
the CD signal at 222 nm as a global probe. Plots of the fraction
denatured versus denaturant concentration obtained from these studies are
identical for all probes under all conditions studied. This observation
provides strong evidence for two-state folding, indicating that there are
no populated intermediates in the folding of lambda 6-85.