Temperature-dependent Dynamics of the Villin Headpiece Helical Subdomain, An Unusually Small Thermostable Protein

Vugmeyster, Liliya; Trott, Oleg; McKnight, C. James; Raleigh, Daniel P.; Palmer, Arthur G.

doi:10.1016/s0022-2836(02)00537-5

Cited by 69 publications

(140 citation statements)

References 46 publications

Supporting

Mentioning

127

Contrasting

Order By: Relevance

“…1). NMR and H/D exchange data had indicated that the GLF-region is flexible in the folded state of HP35, whereas helix 3 is well-defined (16,20,36). According to the X-ray structures, a stable C-terminal helix should prevent contact formation between positions 23 and 35 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

Reiner

Henklein

Kiefhaber

2010

Proc. Natl. Acad. Sci. U.S.A.

120

195

View full text Add to dashboard Cite

A reversible structural unlocking reaction, in which the closepacked van der Waals interactions break cooperatively, has been found for the villin headpiece subdomain (HP35) using triplet-triplet-energy transfer to monitor conformational fluctuations from equilibrium. Unlocking is associated with an unfavorable enthalpy change (ΔH 0 ¼ 35 AE 4 kJ∕mol) which is nearly compensated in free energy by the entropy change (ΔS 0 ¼ 112 AE 20 J · mol −1 · K −1 ). The unlocking reaction has a time constant of about 1 μs at 5°C and is enthalpy-limited with an activation energy of 32 AE 1 kJ∕mol and a large Arrhenius preexponential factor of A ¼ 7.5 × 10 11 s −1 . In the unlocked state a fast local conformational fluctuation with a time constant of 170 ns and a low activation barrier of 17 AE 1 kJ∕mol leads to unfolding of the Cterminal helix and to its undocking from the core. On a much slower time scale, global unfolding occurs from the unlocked state. These results suggest that native protein structures are locked into conformations with low amplitude motions. Large scale motions and global unfolding require an initial structural unlocking step leading to a state with properties of a dry molten globule. The experiments additionally yielded information on the dynamics of loop formation between different positions in unfolded HP35. Comparison of the results with dynamics in unstructured model peptides indicates slightly decelerated kinetics of local loop formation in the Cterminal region which points at residual, nonrandom structure. Dynamics of long-range loop formation, in contrast, are not influenced by residual structure, which argues against unfolded state properties as molecular origin for ultrafast folding of HP35.dry molten globule | native-state fluctuations | protein dynamics | protein folding | unfolded state dynamics N ative states of proteins are known to be close-packed but almost nothing is known about at which stage of the folding process packing occurs. Shaknovich and Finkelstein proposed in 1989 (1) that heat-induced protein unfolding begins with a slight expansion of the protein, such that at least some of the close-packing interactions are broken but water cannot yet penetrate into the interior. This dry molten globule state was postulated to be located on the unfolded side of the major folding barrier but it was argued that it should not be experimentally detectable for water soluble proteins (1). Later, GdmCl-induced unfolding experiments on ribonuclease A (2) and dihydrofolate reductase (DHFR) (3) monitored by time resolved NMR spectroscopy observed rapid formation of an intermediate located on the native side of the major unfolding barrier with properties resembling those of the predicted dry molten globule. The observation of an intact hydrogen-bonding network in the unfolding intermediate of ribonuclease A supported the dry nature of this state (4). Recently, FRET-detected unfolding kinetics of monellin showed rapid expansion of the native state prior to global unfolding (5), which is also in line w...

show abstract

Section: Resultsmentioning

confidence: 99%

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

Reiner

Henklein

Kiefhaber

2010

Proc. Natl. Acad. Sci. U.S.A.

120

195

View full text Add to dashboard Cite

show abstract

“…Additional contributions involving protons at larger distances are minor (the standard deviation across the residues is 3%) and can be neglected. Also, as explained in the Experimental section, the components of 13 C and 15 N CSA tensors were calculated from their isotropic chemical shift values, which already have an implicit averaging over the exchanging sites. We are thus left with the values of the angles in Eqn (5) as the major source of uncertainties.…”

Section: Resultsmentioning

confidence: 99%

“…HP36 has been shown to tumble isotropically, and the values of the molecular tumbling times have been determined over a broad range of temperatures. [13] The tumbling times for 8, 18, and 32…”

Section: Resultsmentioning

confidence: 99%

“…Relaxation dispersion on the multiple quantum C -N coherences would be a logical step to undertake, [20] but the approach has yet to overcome a number of technical challenges. 13 C R 1ρ experiments can be useful in providing further details on microseconds to milliseconds dynamics of HP36's backbone.…”

Section: Resultsmentioning

confidence: 99%

“…[13] Slow microseconds to milliseconds timescale motions, which are most important for many biological functions, [14,15] are much less understood. The existence of slow motions in the folded state of HP36 at temperatures as low as 22 • C has been previously detected by NMR cross-correlated dipole-dipole relaxation of successive amide bonds.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Slow backbone dynamics of chicken villin headpiece subdomain probed by NMR C′N cross‐correlated relaxation

Vugmeyster

2009

Magnetic Reson in Chemistry

Self Cite

View full text Add to dashboard Cite

We have investigated slow correlated motions of neighboring carbonyl and nitrogen nuclei in the backbone of chicken villin headpiece subdomain. Cross-correlated chemical shift modulation experiments were performed at three temperatures where the protein remains in its folded state. The results at 8 degrees C demonstrate the presence of microseconds to milliseconds timescale motions for a number of residues belonging both to helices and unstructured regions. As the temperature is raised, the motions become progressively less visible. The reduction of the contributions of slow motions into the cross-correlated relaxation rate with the rise in temperature is caused by the increase of the chemical exchange rate constants for the slow motion processes.

show abstract

Protein Dynamics

Alpert

Rivet

2011

Encyclopedia of Analytical Chemistry

View full text Add to dashboard Cite

show abstract

Temperature-dependent Dynamics of the Villin Headpiece Helical Subdomain, An Unusually Small Thermostable Protein

Cited by 69 publications

References 46 publications

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

An unlocking/relocking barrier in conformational fluctuations of villin headpiece subdomain

Slow backbone dynamics of chicken villin headpiece subdomain probed by NMR C′N cross‐correlated relaxation

Protein Dynamics

Contact Info

Product

Resources

About