Backbone‐Only Protein Solution Structures with a Combination of Classical and Paramagnetism‐Based Constraints: A Method that Can Be Scaled to Large Molecules

Abstract

Herein, it is shown that a medium-resolution solution structure of a protein can be obtained with the sole assignment of the protein backbone and backbone-related constriants if a derivative with a firmly bound paramagnetic metal is available. The proof-of-concept is provided on calbindin D9k, a calcium binding protein in which one of the two calcium ions can be selectively substituted by a paramagnetic lanthanide ion. The constraints used are HN (and Ha) nuclear Overhauser effects (NOEs), hydrogen bonds, dihedral angle constriants from chemical shifts, and the following paramagnetism-based constraints: 1) pseudocontact shifts, acquired by substituting one (or more) lanthanide(s) in the C-terminal calcium binding site; 2) N-HN residual dipolar couplings due to self-orientation induced by the paramagnetic lanthanide(s); 3) cross-correlations between the Curie and internuclear dipole-dipole interactions; and 4) paramagnetism-induced relaxation rate enhancements. An upper distance limit for internuclear distances between any two backbone atoms was also given according to the molecular weight of the protein. For this purpose, the paramagnetism-based constraints were collectively implemented in the program CYANA for solution structure determinations, similarly to what was previously done for the program DYANA. The method is intrinsically suitable for large molecular weight proteins.

NMR Spectroscopy of Paramagnetic Metalloproteins

“…It was also shown that paramagnetism-based restraints, together with identification of the secondary structure elements, may provide the backbone solution structure of a metalloprotein even in the absence of any NOEs. [64,94,108] In conclusion, paramagnetism-based restraints have been proven to be precious for the solution structure determination of paramagnetic proteins. This is particularly true if, for any reason, the number of NOEs is limited.…”

“…However, it was shown that the d pcs values are absolutely consistent with NOEs and dihedral-angle structural restraints. [60][61][62] A protocol has been thus implemented to include these restraints in the most popular software packages for solution structure determination, such as DYANA/ CYANA [63,64] and Xplor-NIH. [65] Although such programs are not optimized for handling these restraints, due to the complicated form of the energy surface, they have been shown to be precious not only for protein refinement but also for ab initio structural calculations.…”

“…[65] Although such programs are not optimized for handling these restraints, due to the complicated form of the energy surface, they have been shown to be precious not only for protein refinement but also for ab initio structural calculations. [64] In general, the d pcs values can be measured after the complete assignment of the 1 H, 15 N-HSQC spectra of both the diamagnetic and the paramagnetic samples is obtained. However, when the assignment of the diamagnetic spectrum is available, a shortcut is possible.…”

“…Protocols for the use of Curie dipolar relaxation cross-correFlation rate restraints (ccrs) are available at the web site www.postgenomicnnmr.net for the programs PARAMAGNETIC DYANA/CYANA [64,90] and Xplor-NIH. [65] Paramagnetic cross-correlation effects were first discussed in 1993, [91,92] measured for HN metalloproteins in 1999, [93] and have been used as restraints since 2000.…”

Paramagnetic shifts in solid-state NMR of proteins to elicit structural information

“…1) were used (47). ¶ Such calculations also show that some distance restraints are always needed, otherwise pcs alone hardly converge to a correct folding (22,48).…”