It and averaging as reported by scalar couplings. For example, T39 is far from statistics and tR,eff values below 3 ns is quite sensi- the site of mutation in F22L. The system-dependent. An exact knowledge of A is not increase in the rate of T39 rotameric averaging in required to assess the kinetic effects of changes in F22L may therefore be due to slight changes barrier height that result from a point mutation, in tertiary structure relative to A20V and WT that for example, so long as it does not change between disrupt this interaction, by increasing either the the variants, since: Hg1 — O separation or the Og1 — Hg1 — O angle.
As described above, in the case of surface residue depend to a large extent upon its protein L simulations have established that the intra-residue backbone conformation33 and it may 2 H spin relaxation data are sensitive to slow time- be that the energetic barriers separating rotameric scale dynamics extending from approximately states are also sensitive to backbone geometry. The 1 —10 ns so long as tR,eff , 3 ns. Thus, for T39 A20V and WT X-ray structures establish that there where nanosecond dynamics are observed only in is a greater than difference in the c dihedral F22L and where averaging about x1 does take angle of T19 in the two forms of the protein, place in all three variants established by the scalar which may well affect the x1 dynamics of this couplings , the motional time-scale must change by residue.
T19 is not the only residue to show signifi- at least an order of magnitude between F22L and cant changes in local backbone conformation.
Six the other two forms of the protein. It is likely, residue. The order parameter of L40d1, how- residues in a 50 ns molecular dynamics ever, is the same in all three proteins, suggesting simulation of the N-terminal SH3 domain from that the additional dynamics detected by L40d2 are drk are in good agreement with equation 3 , highly anisotropic, occurring mainly about an axis using barrier heights from the trajectory and with of rotation parallel with the Cg — Cd1 bond vector.
A set to the Eyring prefactor. Although these conformational changes are quite minor, the structural determinants of fast time-scale side- Conclusions chain motions are not well understood,34 and local structural rearrangement likely mediates the effect Here, we have used newly developed 2H spin of the A20V mutation on L40 dynamics.
Similarly relaxation measures in concert with three-bond small structural perturbations may be responsible scalar coupling data to investigate how the for the nanosecond time-scale motions detected dynamics of side-chain methyl groups change in for L10 only in F22L and not in A20V or WT. The increase in stability of the A20V mutant may be completely rationalized Analysis in terms of energetic barriers in terms of the hydrophobicity difference between alanine and valine residues, and its structure is Changes in local energetics associated with shifts essentially identical with that of the WT protein.
In contrast, the destabilization of vectors in the 1HZ6 structure. Local diffusion constants F22L is significantly greater than what would be were calculated from 15N R1 and R1r relaxation rates and predicted from the modest difference in hydro- fit to an axially symmetric global diffusion tensor using phobicity between phenylalanine and leucine the program quadric. Here too, however, dipolar couplings tumbling were not justified on the basis of F-test statis- establish that at least globally the structure does tics.
The relaxation rates obtained from the five 2H not change. Since there is a greater potential for relaxation experiments were shown to be self-consistent disruption of specific favorable interactions in the using methods described previously.
Nonetheless, as et al. Notably, many of the residues affected are tation of the Cmethyl — C axis with respect to the diffusion quite distant. The LS-2 of long-range conformational coupling to F Deuterium relaxation data were cooperative nature of protein folding.
This study additionally fit using the same equations with the corre- establishes the utility of 2H spin relaxation experi- lation time optimized on a per methyl group basis ments to detect and characterize motions on the LS-3 yielding residual x2LS3 values resultant spectral nanosecond and picosecond time-scales.
The density functions shown in red. The five 2H relaxation rates were also used to calculate unique sets of three spectral density function internal motions in proteins. To obtain Escherichia coli BL21 DE3 cells were used to obtain ranges of tR,eff values consistent with the absence of protein samples. Partial alignment of samples was achieved of S 2 and te varying uniformly from 0.
Random noise with a Gaussian and leucine were obtained using the method of Neri profile was added to the simulated data using standard et al. The resulting rates were subse- Data analysis and model selection quently fit with tR,eff included as an adjustable parameter replacing tR i.
LS-3 model. The process was repeated Parameters for each molecule describing the magni- , times to generate the distribution shown in tude Aa , anisotropy Ar and orientation a,b,g of Figure 3B. Deuterium spin probes of side- detected from fits of data involving the LS-3 model chain dynamics in proteins.
Measurement of five additional data sets were simulated using the LS-4 relaxation rates per deuteron in 13C-labeled and spectral density equation 2 , with 0. The data Chem. Skrynnikov, N. Spectral density mapping and identifi- obtained Figure 3C and D : , simulations were cation of nanosecond time-scale side-chain motions. Scalley, M. Kinetics of folding of the IgG binding domain of peptostreptococcal protein L. Biochemistry, 36, — Acknowledgements Kim, D.
The sequences of small proteins are not extensively optimized for This work was supported by a grant from the rapid folding by natural selection. Natl Acad. Canadian Institutes of Health Research. USA, 95, — Fauchere, J. Hydrophobic parameters pi of amino acid side chains from the L.
The authors thank Dr Regis Pomes J. Hospital for Sick Children, Toronto for useful Lee, L. Rotational diffusion anisotropy of proteins 50 ns molecular dynamics trajectory of an SH3 from simultaneous analysis of 15N and 13Ca nuclear domain and Dr Rhea Hudson Hospital for Sick spin relaxation. NMR, 9, — Children, Toronto for help with cloning. Bowie, J. Deciphering the message in protein sequences. Science, , — Eriksson, A.
Brannigan, J. Protein Response of a protein structure to cavity- engineering 20 years on. Nature Rev. Cell Biol. Wand, A. Dynamic activation of protein Mulder, F. Dahlquist, F. Flexibility and Nature Struct. Eisenmesser, E. Biochemistry, Enzyme dynamics during catalysis. Science, 39, — Johnson, E. Hammes-Schiffer, S. Impact of enzyme Handel, T.
Solution structure and motion on activity. Biochemistry, 41, — Ishima, R. Protein dynamics ubiquitin. Structure, 7, — Nature Struct. Lipari, G. Model-free approach to 6. Kay, L. Backbone the interpretation of nuclear magnetic relaxation in dynamics of proteins as studied by 15N inverse macromolecules: 2.
Analysis of experimental results. Biochemistry, 28, Model-free approach to — Protein dynamics from NMR. Theory and range of validity.
NMR, 5 suppl. Choy, W. Model selection for S. Correlation between the interpretation of protein side chain methyl binding and dynamics at SH2 domain interfaces. In the press. Clore, G.
Mittermaier, A. Deviations from L. Changes in side chain and the simple 2 parameter model free approach to the backbone dynamics identify determinants of speci- interpretation of 15N nuclear magnetic relaxation of ficity in RNA recognition by human U1A protein.
Zar, J. Two-sample hypotheses. In Biostatis- Lee, A. Microscopic origins tical Analysis, pp. Nature, , — Bax, A. Measurement of Walsh, S. Dynamics of a de novo designed three- peptide complex. Vuister, G. NMR relaxation methods. Biochemistry, 40, Measurement of three-bond nitrogen— carbon J coup- — Millet, O. Wang, A. Measure- Nature Struct. Ottiger, M. Tuesday 10 August Wednesday 11 August Thursday 12 August Friday 13 August Saturday 14 August Sunday 15 August Monday 16 August Tuesday 17 August Wednesday 18 August Thursday 19 August Friday 20 August Saturday 21 August Sunday 22 August Monday 23 August Tuesday 24 August Wednesday 25 August Thursday 26 August Friday 27 August Saturday 28 August Sunday 29 August Monday 30 August Tuesday 31 August Wednesday 1 September Thursday 2 September Friday 3 September Saturday 4 September Sunday 5 September Monday 6 September Tuesday 7 September Wednesday 8 September Thursday 9 September Friday 10 September Saturday 11 September Sunday 12 September Monday 13 September Tuesday 14 September Wednesday 15 September Thursday 16 September Friday 17 September Saturday 18 September Sunday 19 September Monday 20 September Tuesday 21 September Wednesday 22 September Thursday 23 September Friday 24 September Saturday 25 September Sunday 26 September Monday 27 September Tuesday 28 September Wednesday 29 September Thursday 30 September Friday 1 October Saturday 2 October Sunday 3 October Monday 4 October Tuesday 5 October Wednesday 6 October Thursday 7 October Friday 8 October Saturday 9 October Sunday 10 October Monday 11 October Tuesday 12 October Wednesday 13 October Thursday 14 October Friday 15 October Saturday 16 October Sunday 17 October Monday 18 October Tuesday 19 October Wednesday 20 October Thursday 21 October Friday 22 October Saturday 23 October Sunday 24 October Monday 25 October Tuesday 26 October Wednesday 27 October Thursday 28 October Friday 29 October Saturday 30 October Sunday 31 October Monday 1 November Tuesday 2 November Wednesday 3 November Thursday 4 November Friday 5 November Saturday 6 November Sunday 7 November Monday 8 November Tuesday 9 November Wednesday 10 November Thursday 11 November Friday 12 November Saturday 13 November Sunday 14 November Monday 15 November Tuesday 16 November Wednesday 17 November Thursday 18 November Friday 19 November Saturday 20 November Sunday 21 November Monday 22 November Tuesday 23 November Wednesday 24 November Thursday 25 November Friday 26 November Saturday 27 November Sunday 28 November
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