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Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins.

ABSTRACT: The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve (15)N and (1)HN resonances. The assignment becomes problematic when there is resonance overlap of (15)N-(1)HN cross peaks. For such residues, one cannot unambiguously link the "left" side of the NH root to the "right" side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a solution to this problem: a hybrid (4d,3d) reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance is modulated with the frequency of the Ca(i-1) resonance, which helps in resolving the ambiguity involved in connecting the Ca(i) and Ca(i-1) resonances for overlapping NH roots. The experiment has limited sensitivity, and is only suited for small or unfolded proteins. In a companion experiment, (4d,3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(i-1) resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(i-1) resonances for overlapping NH roots.

SUBMITTER: Bagai I

PROVIDER: S-EPMC3091507 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins.

Bagai Ireena I Ragsdale Stephen W SW Zuiderweg Erik R P ER

Journal of biomolecular NMR 20101230 2

The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve (15)N and (1)HN resonances. The assignment becomes problematic when there is resonance overlap of (15)N-(1)HN cross peaks. For such residues, one cannot unambiguously link the "left" side of the NH root to the "right" side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a solution to this problem: a hy ...[more]

PMID: 21190062

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Project description:Experimental residual dipolar couplings (RDCs) in combination with structural models have the potential for accelerating the protein backbone resonance assignment process because RDCs can be measured accurately and interpreted quantitatively. However, this application has been limited due to the need for very high-resolution structural templates. Here, we introduce a new approach to resonance assignment based on optimal agreement between the experimental and calculated RDCs from a structural template that contains all assignable residues. To overcome the inherent computational complexity of such a global search, we have adopted an efficient two-stage search algorithm and included connectivity data from conventional assignment experiments. In the first stage, a list of strings of resonances (CA-links) is generated via exhaustive searches for short segments of sequentially connected residues in a protein (local templates), and then ranked by the agreement of the experimental (13)C(α) chemical shifts and (15)N-(1)H RDCs to the predicted values for each local template. In the second stage, the top CA-links for different local templates in stage I are combinatorially connected to produce CA-links for all assignable residues. The resulting CA-links are ranked for resonance assignment according to their measured RDCs and predicted values from a tertiary structure. Since the final RDC ranking of CA-links includes all assignable residues and the assignment is derived from a "global minimum", our approach is far less reliant on the quality of experimental data and structural templates. The present approach is validated with the assignments of several proteins, including a 42 kDa maltose binding protein (MBP) using RDCs and structural templates of varying quality. Since backbone resonance assignment is an essential first step for most of biomolecular NMR applications and is often a bottleneck for large systems, we expect that this new approach will improve the efficiency of the assignment process for small and medium size proteins and will extend the size limits assignable by current methods for proteins with structural models.

Dataset Information

Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins.

Publications

Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins.

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