Polar Versus Non-polar Local Ordering at Mobile Sites in Proteins: Slowly Relaxing Local Structure Analysis of (15)N Relaxation in the Third Immunoglobulin-Binding Domain of Streptococcal Protein G.

Abstract

We developed recently the slowly relaxing local structure (SRLS) approach for studying restricted motions in proteins by NMR. The spatial restrictions have been described by potentials comprising the traditional L = 2, K = 0, 2 spherical harmonics. However, the latter are associated with non-polar ordering whereas protein-anchored probes experience polar ordering, described by odd-L spherical harmonics. Here we extend the SRLS potential to include the L = 1, K = 0, 1 spherical harmonics and analyze (15)N-(1)H relaxation from the third immunoglobulin-binding domain of streptococcal protein G (GB3) with the polar L = 1 potential (coefficients c0(1) and c1(1)) or the non-polar L = 2 potential (coefficients c0(2) and c2(2)). Strong potentials, with ⟨c0(1)⟩ ∼ 60 for L = 1 and ⟨c0(2)⟩ ∼ 20 for L = 2 (in units of kBT), are detected. In the α-helix of GB3 the coefficients of the rhombic terms are c1(1) ∼ c2(2) ∼ 0; in the preceding (following) chain segment they are ⟨c1(1)⟩ ∼ 6 for L = 1 and ⟨c2(2)⟩ ∼ 14 for L = 2 (⟨c1(1)⟩ ∼ 3 for L = 1 and ⟨c2(2)⟩ ∼ 7 for L = 2). The local diffusion rate, D2, lies in the 5 × 10(9)-1 × 10(11) s(-1) range; it is generally larger for L = 1. The main ordering axis deviates moderately from the N-H bond. Corresponding L = 1 and L = 2 potentials and probability density functions are illustrated for residues A26 of the α-helix, Y3 of the β1-strand, and L12 of the β1/β2 loop; they differ considerably. Polar/orientational ordering is shown to be associated with GB3 binding to its cognate Fab fragment. The polarity of the local ordering is clearly an important factor.