NMR Solution Structure of the Isolated N-Terminal Fragment of Protein-G B1 Domain. Evidence of Trifluoroethanol Induced Native-Like .beta.-Hairpin Formation

Fj, Blanco; Ma, Jiménez; Pineda, Agustin O.; Rico, Manuel; Santoro, Jorge; Jl, Nieto

doi:10.1021/bi00185a041

Cited by 188 publications

(137 citation statements)

References 79 publications

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“…At this temperature, hairpin 2 is Ϸ40% native, whereas the helix and hairpin 1 are negligibly stable. This finding agrees with experiments (28,29) that have shown that hairpins 1 and 2 are Ϸ0% and Ͻ42% stable (at pH 6.3, 278 K), and with an independent empirical calculation (30) that predicts Ͻ10% stability for the helix (at pH 7.0, 278 K).…”

supporting

confidence: 91%

The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation

Shimada

Shakhnovich

2002

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

171

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Protein G is folded with an all-atom Monte Carlo simulation by using a Gō potential. When folding is monitored by using burial of the lone tryptophan in protein G as the reaction coordinate, the ensemble kinetics is single exponential. Other experimental observations, such as the burst phase and mutational data, are also reproduced. However, more detailed analysis reveals that folding occurs over three distinct, three-state pathways. We show that, because of this tryptophan's asymmetric location in the tertiary fold, its burial (i) does not detect certain intermediates and (ii) may not correspond to the folding event. This finding demonstrates that ensemble averaging can disguise the presence of multiple pathways and intermediates when a non-ideal reaction coordinate is used. Finally, all observed folding pathways eventually converge to a common rate-limiting step, which is the formation of a specific nucleus involving hydrophobic core residues. These residues are conserved in the ubiquitin superfamily and in a phage display experiment, suggesting that fold topology is a strong determinant of the transition state.B alancing realism and computational tractability has been the central issue when simulating protein folding on the computer. Recently, an impressive parallelization effort led to a 1-s full-scale molecular dynamics (MD) trajectory of the 36-residue villin headpiece (1). Unfortunately, the use of such simulations to rigorously study folding is still several years away because single domain proteins fold on timescales that are at least two orders of magnitude longer (2). Moreover, because folding is a stochastic process, averaging over multiple runs is required. These computational problems can be partly alleviated by investigating folding kinetics indirectly by using the construction of free energy landscapes (3) or unfolding at high temperatures (4).Recently, two complementary approaches have directly accessed the timescales relevant to folding. The first makes use of ensemble dynamics (5), whereby the long waiting times associated with rare events-which plague any simulation being run serially in time-are eliminated by running parallel simulations and allowing them to exchange states whenever a barrier crossing occurs. The second approach extends existing off-lattice coarsegrained Monte Carlo (MC) simulations (6) by introducing all-atom structural realism. Computational costs are minimized by (i) moving only backbone and sidechain torsional degrees of freedom (which are the ''softest'' modes in a polymer) and (ii) by using coarse-grained potentials. This simulation has been used with the Gō (7) and sequence-based potentials (8) to fold helices, hairpins, crambin, and protein A.In this paper, we present ensemble kinetic data of the wellcharacterized 57-residue protein G (9) (Fig. 1A) by using this all-atom MC technique with a Gō potential. Under this potential, only interactions present in the native conformation are attractive. Although the native state is the global energy minimum by construction, no...

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supporting

confidence: 91%

The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation

Shimada

Shakhnovich

2002

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

171

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“…This trace was then complex FFT and the separation between the antiphase components was taken to be the coupling constant. For a few peaks, sine-squared window function shifted by 308 was applied when a stronger window function led to distortion of the peaks 1 H-13 C Gradient-heteronuclear single quantum correlation (HSQC) [24] experiments with sensitivity enhancement were recorded at 0, 27, 36, 45, 54, 63 and 90% TFE to monitor the changes in 13 Ca chemical shifts. Spectral width used for the F1 dimension was 12 000 Hz and 16 scans were added for each of the 600 t 1 increments.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

CD and NMR investigations on trifluoroethanol‐induced step‐wise folding of helical segment from scorpion neurotoxin

Khandelwal

Seth

Hosur

1999

European Journal of Biochemistry

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A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly a-helix covering all of the 14 residues has been studied by 1 H and 13 C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the stepwise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5±11 even at low TFE concentrations. It was also observed that . 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.

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“…Peptide fragments of GB1 have also been studied in isolation in water and trifluoroethanol, and it has been shown that a peptide comprising the ~3-]34 turn exhibits a significant population of native conformer in water [28,29].…”

Section: Introductionmentioning

confidence: 99%

Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity

1996

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A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H-lSN heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in ~-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the ~-sheet side of the sheet-helix core interface. A solvent exposed position Ile-6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume.

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NMR Solution Structure of the Isolated N-Terminal Fragment of Protein-G B1 Domain. Evidence of Trifluoroethanol Induced Native-Like .beta.-Hairpin Formation

Cited by 188 publications

References 79 publications

The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation

The ensemble folding kinetics of protein G from an all-atom Monte Carlo simulation

CD and NMR investigations on trifluoroethanol‐induced step‐wise folding of helical segment from scorpion neurotoxin

Core mutants of the immunoglobulin binding domain of streptococcal protein G: Stability and structural integrity

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