Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Jan 30;135(4):1415-22.
doi: 10.1021/ja309527h. Epub 2013 Jan 22.

Folding and binding of an intrinsically disordered protein: fast, but not 'diffusion-limited'

Joseph M Rogers  1 , Annette Steward, Jane Clarke
Affiliations

Folding and binding of an intrinsically disordered protein: fast, but not 'diffusion-limited'

Joseph M Rogers et al. J Am Chem Soc. .

Abstract

Coupled folding and binding of intrinsically disordered proteins (IDPs) is prevalent in biology. As the first step toward understanding the mechanism of binding, it is important to know if a reaction is 'diffusion-limited' as, if this speed limit is reached, the association must proceed through an induced fit mechanism. Here, we use a model system where the 'BH3 region' of PUMA, an IDP, forms a single, contiguous α-helix upon binding the folded protein Mcl-1. Using stopped-flow techniques, we systematically compare the rate constant for association (k(+)) under a number of solvent conditions and temperatures. We show that our system is not 'diffusion-limited', despite having a k(+) in the often-quoted 'diffusion-limited' regime (10(5)-10(6) M(-1) s(-1) at high ionic strength) and displaying an inverse dependence on solvent viscosity. These standard tests, developed for folded protein-protein interactions, are not appropriate for reactions where one protein is disordered.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A) Cartoon depicting Mcl-1 (gray) binding PUMA peptide (blue). Unbound Mcl-1 is based on pdb 1WSX, ensemble of structures of unbound PUMA peptide built using Chimera (UCSF) and bound structure is based on pdb 2ROC. Figure prepared using PyMol. (B) Full-length PUMA is predicted to be entirely disordered, producing a PONDR-FIT score 0.5–1.0, and has residual helicity only in the BH3 region used in this study (magenta), as predicted by the helical propensity predictor AGADIR. (C) Consistent with a coupled folding and binding reaction, PUMA peptide binds Mcl-1 with an increase in helicity, as shown by circular dichroism. The 1:1 complex (black solid line) has a greater α-helical signal than the spectrum predicted for no interaction (dashed line), which is the sum of the PUMA alone (blue) and Mcl-1 alone (magenta) spectra. (D) Kinetics of association between Mcl-1 and PUMA peptide could be followed by stopped-flow fluorescence. An increase in temperature accelerates association. Fits for irreversible association (eq 3) are shown as black lines.
Figure 2
Figure 2
(A) Association is partly driven by electrostatic interactions between PUMA and Mcl-1 and k+ is highly dependent on the ionic strength of the solution. Where repeat measurements were made standard errors are shown as error bars. The fit of the data to eq 4 is shown (black line) (see also Figure S7). No buffer specific effects are seen as the rate constant in the standard biophysics buffer (50 mM PO4, pH 7, I = 109 mM, ○) matches that in the MOPS buffer with I corrected using NaCl (●). (B) k+ is highly dependent on the solvent viscosity; there is good agreement between the experimental k+ (●) and the predicted k+ (k+ = k0η0/η, ○). (C) The standard viscosity plot for relative rate constant vs relative viscosity has a slope close to unity 1.13 ± 0.02 (shown as a black line), for comparison a slope of 1 is shown as a dashed line.
Figure 3
Figure 3
(A) The experimental association rate constant k+ (●) is not directly proportional to T/η, as would be predicted for a ‘diffusion-limited’ reaction according to eq 2 (○, arbitrarily drawn through the 10 °C data point). Where repeat measurements were made, standard errors are shown as error bars. (B) Temperature corrected viscosity plot shows clear nonlinearity, in contrast to what is expected for a ‘diffusion-limited’ reaction (gradient of 1 shown as a dashed line). (C) k+ is reduced by increasing the concentration of the denaturant urea (●) and this effect is not accounted for by the slower diffusion due to changes in solvent viscosity (k+ = k0η0/η, ○). (D) The log of the rate constant is linearly dependent on urea concentration, suggesting that a structured state is being energetically disfavored.
Figure 4
Figure 4
(A) Cartoon depicting Mcl-1 (gray) binding GB1-PUMA peptide (green). Unbound Mcl-1 is based on pdb 1WSX, cartoon of unbound GB1-PUMA peptide is based on pdb 3GB1, built using Chimera (UCSF), and bound structure is based on pdb 2ROC. Figure prepared using PyMol. (B) The rate constant for GB1-PUMA (green □) Mcl-1 association has a very similar temperature dependence to the smaller PUMA peptide (●). Where repeat measurements were made standard errors are shown as error bars. (C) GB1-PUMA (green □) k+ is very dependent on ionic strength and shows very similar behavior to the PUMA peptide (black circles). The fit to the data shown here (black line) is obtained from a plot of ln(k+) vs 1/(1 + κa) (Figure S7, Methods).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wright P. E.; Dyson H. J. J. Mol. Biol. 1999, 293, 321. - PubMed
    1. Uversky V. N.; Dunker A. K. Biochim. Biophys. Acta 2010, 1804, 1231. - PMC - PubMed
    1. Ward J. J.; Sodhi J. S.; McGuffin L. J.; Buxton B. F.; Jones D. T. J. Mol. Biol. 2004, 337, 635. - PubMed
    1. Dunker A. K.; Brown C. J.; Lawson J. D.; Iakoucheva L. M.; Obradovic Z. Biochemistry 2002, 41, 6573. - PubMed
    1. Wright P. E.; Dyson H. J. Curr. Opin. Struct. Biol. 2009, 19, 31. - PMC - PubMed

Publication types