Characterization of Large Peptide Fragments Derived from the N-Terminal Domain of the Ribosomal Protein L9:  Definition of the Minimum Folding Motif and Characterization of Local Electrostatic Interactions

Abstract

A set of peptides derived from the N-terminal domain of the ribosomal protein L9 (NTL9) have been characterized in an effort to define the minimum unit of this domain required to fold and to provide model peptides for the analysis of electrostatic interactions in the unfolded state. NTL9 is a 56-residue alpha-beta protein with a beta1-loop-beta2-alpha1-beta3-alpha2 topology. The beta-sheet together with the first helix comprise a simple example of a common supersecondary motif called the split beta-alpha-beta fold. Peptides corresponding to the beta1-loop-beta2 unit are unstructured even when constrained by an introduced disulfide. The pK(a)s of Asp-8 and Glu-17 in these peptides are slightly lower than the values found for shorter peptides but are considerably higher than the values in NTL9. A 34-residue peptide, which represents the beta1-loop-beta2-alpha1 portion of NTL9, is also unstructured. In contrast, a 39-residue peptide corresponding to the entire split beta-alpha-beta motif is folded and monomeric as judged by near- and far-UV CD, two-dimensional NMR, ANS binding experiments, pK(a) measurements, and analytical ultracentrifugation. The fold is very similar to the structure of this region in the intact protein. Thermal and urea unfolding experiments show that it is cooperatively folded with a DeltaG degrees of unfolding of 1.8-2.0 kcal/mol and a T(m) of 58 degrees C. This peptide represents the first demonstration of the independent folding of an isolated split beta-alpha-beta motif, and is one of only four naturally occurring sequences of fewer than 40 residues that has been shown to fold cooperatively in the absence of disulfides or ligand binding.

Electrostatic interactions in the denatured state ensemble: Their effect upon protein folding and protein stability

“…Peptide fragment studies rule out the possibility that the effects arise from a propensity for the local sequence to encode a preferred conformation which leads to a strong interaction between D8 and K12. A peptide fragment consisting of the first 22 residues of NTL9, which included the β-strand -1, loop, β strand -2 unit, was not able to fold in isolation [37] even when the N and C-termini were constrained by a Gly-Cys-CysGly linker. Constraining the fragment had only modest effects on the pK a of D8 or E17.…”

Nascent β -Hairpin Formation of a Natively Unfolded Peptide Reveals the Role of Hydrophobic Contacts

“…Circular dichroism and NMR-based pK a measurements were conducted on several fragment peptides of NTL9, including residues 1-11 and 1-22. It was found that even though both fragments are unstructured, the pK a of Asp 8 is shifted lower than the model value (4.0) by 0.16 and 0.34 units, respectively (13,14). Interestingly, these shifts are slightly smaller than the pK a of Asp 8 in a destabilizing mutant, V3AI4A, which was estimated to be 0.44 units (21).…”

“…We note that the unfolded states are also referred to by some workers as denatured states, since historically they were obtained using chemical denaturants or acids. The unfolded states have been traditionally thought of as random coils without preferential interactions (5,6); however, an increasing number of experimental studies point to the presence of residual structures such as the native-like topology, local and longrange order (7)(8)(9), as well as specific hydrophobic (10)(11)(12) and electrostatic interactions (13)(14)(15).…”

“…The minimum folding unit includes the first 39 residues and lacks the second helix (14). Thus, adding the third b-strand along with the intervening helix stabilizes the b-hairpin, b1-loop-b2, which is unstructured on its own (14). This b-hairpin, corresponding to residues 1-22, is the focus of this article.…”

cDNA, genomic sequence cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda (Ailuropoda melanoleuca)

“…Previous study has shown that L9 is an elongated protein with a 34-residue α-helix that connects and orients the N-(NTL9) and C-terminal (CTL9) domains, which both contain a predicted RNA-binding site (Adamski et al, 1996;Lieberman et al, 2000). NTL9 is a 56-residue α-b globular protein with a b1-loop-b2-α1-b3-α2 topology (Horng et al, 2002), and CTL9 is a 92-residue α-b globular protein containing two α-helices and an unusual three-stranded mixed parallel, anti-parallel b-sheet (Hoffman et al, 1996).…”