Visualizing the principal component of 1H,15N-HSQC NMR spectral changes that reflect protein structural or functional properties: application to troponin C

Abstract

Laboratories often repeatedly determine the structure of a given protein under a variety of conditions, mutations, modifications, or in a number of states. This approach can be cumbersome and tedious. Given then a database of structures, identifiers, and corresponding (1)H,(15)N-HSQC NMR spectra for homologous proteins, we investigated whether structural information could be ascertained for a new homolog solely from its (1)H,(15)N-HSQC NMR spectrum. We addressed this question with two different approaches. First, we used a semi-automated approach with the program, ORBplus. ORBplus looks for patterns in the chemical shifts and correlates these commonalities to the explicit property of interest. ORBplus ranks resonances based on consistency of the magnitude and direction of the chemical shifts within the database, and the chemical shift correlation of the unknown protein with the database. ORBplus visualizes the results by a histogram and a vector diagram, and provides residue specific predictions on structural similarities with the database. The second method we used was partial least squares (PLS), which is a multivariate statistical technique used to correlate response and predictor variables. We investigated the ability of these methods to predict the tertiary structure of the contractile regulatory protein troponin C. Troponin C undergoes a closed-to-open conformational change, which is coupled to its function in muscle. We found that both ORBplus and PLS were able to identify patterns in the (1)H,(15)N-HSQC NMR data from different states of troponin C that correlated to its conformation.

Structural and Functional Consequences of the Cardiac Troponin C L48Q Ca2+-Sensitizing Mutation

“…Our MD simulations predicted a decrease of approximately 10° in the AB interhelical angle of cNTnC(L48Q)•Ca 2+ . This magnitude of change is consistent with a recent study that used couple of different computational methods in combination with NMR data to predict that the AB angle of cNTnC(L48Q)•Ca 2+ would go from ~130 to ~120° (50). This structural impact is most likely the result of changing the hydrophobic Leu to the hydrophilic Gln at position 48 at the end of the B-helix.…”

Structures reveal details of small molecule binding to cardiac troponin

“…Chemical shift mapping of 1 H and 15 N signals do not seem to accurately delineate the small molecule binding site in cNTnC. Instead, they are sensitive markers of conformational changes in the protein[41] that are needed to accommodate drug binding. The largest 15 N chemical shift changes are seen in Val64 and Met81 in cNTnC, and Ala150 in cTnI, highlighting hotspots of conformational change that occur upon 3-mDPA binding.…”

The structural and functional effects of the familial hypertrophic cardiomyopathy-linked cardiac troponin C mutation, L29Q

“…χ 1 angles were assigned as being either − 60 ± 60°, 180 ± 60°, or 60 ± 60°. Interhelical angles for cNTnC(L29Q) were predicted using ORBplus [48] .…”