NMR Structure Determination for Larger Proteins Using Backbone-Only Data

Abstract

Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without NMR information on the sidechains for proteins up to 25 kDa by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modelling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search towards the lowest energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kDa, and should enable routine NMR structure determination for larger proteins.The first step in protein structure determination by NMR is chemical shift assignment for the backbone atoms. In contrast to the subsequent assignment of the sidechains, this is now rapid, reliable, and largely automated (1-5). Global backbone structural information complementing the local structure information provided by backbone chemical shift assignments (6,7), can be obtained from H N -H N NOESY, residual dipolar coupling (RDC) (8), and other (9,10) experiments. For larger proteins, deuteration becomes necessary to circumvent the efficient spin relaxation properties resulting from their higher rotational correlation times (11,12), but removing protons also eliminates long range NOESY information from sidechains except for selectively protonated sidechain moieties (13). The difficulty in determining accurate structures with no or limited sidechain information is a # To whom correspondence should be addressed. Phone: (206) 543-1295, Fax: (206) Here we show that structures of proteins up to 200 residues (23 kDa) can be determined using information from backbone (H N , N, C α , C β , C') NMR data by taking advantage of the conformational sampling and all atom energy function in the Rosetta structure prediction methodology, which for small proteins in favorable cases can produce atomic accuracy models starting from sequence information alone(15). Structure prediction in Rosetta proceeds in two steps; first a low resolution exploration phase using Monte-Carlo fragment assembly and a coarse-grained energy function, and second a computationally expensive refinement phase which cycles between combinatorial sidechain optimization and gradientbased minimization of all torsional degrees of freedom in a physically-realistic all-atom forcefield(15). The primary obstacle to Rosetta structure prediction from amino acid sequence information alone is conformational sampling; native structures almost always have lower energies than non-native conformations, but are very seldom sampled in unbiased trajectories. Incorpora...

Probing the self‐association, intermolecular contacts, and folding propensity of amelogenin

“…28,29 A direct overlay comparison of the 15 N-1 H HSQC AQ-and 70-rP172 spectra reveals resonant frequency mismatch arising from TFE solvent-exchange-and conformationally induced chemical shift effects. [28][29][30][31][32][33][34][35] Given that monomeric rP172 exists in a globally extended conformational state, 9 it is not surprising that this protein is susceptible to TFE -OH proton exchange [28][29][30] at NH backbone and sidechain sites. As a result, TFE and water proton chemical exchange significantly contribute to the differences in 1 H frequency dispersion [ Fig.…”

“…Since aNH and 15 Na chemical shifts encode solventrelated effects, 10,11,34,35 we analyzed sequence-specific backbone 13 C chemical shift information (i.e., 13 Ca, 13 Cb, 13 CO) to identify regions which experience TFE-induced folding. We performed two analyses.…”

“…For the 70-rP172 sample, protein backbone sequential assignments were obtained using an array of multidimensional NMR experiments (HSQC, HNCA, HNCO, HNCACB, HNCACO, HNCOCA). [9][10][11]35 For the AQ-rP172 sample, 15 N-1 H-HSQC experiments were performed to qualitatively compare the 1 H and 15 N NMR backbone resonance dispersion 34 relative to the 70-rP172 sample and to previously published NMR datasets obtained for monomeric AQ-rP172. 9 NMRPipe software (National Institute of Health, Bethesda, MD) was used to process all NMR data and NMRViewJ (version 8.0.b21 with Java 1.6.0_17, One Moon Scientific, Newark, NJ) was used for sequential assignments.…”

Cross-Validation of the Structure of a Transiently Formed and Low Populated FF Domain Folding Intermediate Determined by Relaxation Dispersion NMR and CS-Rosetta

“…as RDCs can be incorporated to improve structure quality [48][49][50][51]. Typically a protocol is used whereby small fragments are selected from a database of structures based on agreement with experimental data, with models derived subsequently from fragment assembly.…”

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“…In some cases, such as large proteins, additional restraints cannot be obtained due to limitations of spectral quality [32]. In such cases, as well as in molecular modeling and protein design applications, the properties of the conformational ensemble are expected to be sensitive to the…”