Differential Ordering of the Protein Backbone and Side Chains during Protein Folding Revealed by Site-Specific Recombinant Infrared Probes

Abstract

The timescale for ordering of the polypeptide backbone relative to the side chains is a critical issue in protein folding. The interplay between ordering of the backbone and side chains is particularly important for the formation of β-sheet structures, as the polypeptide chain searches for the native stabilizing cross-strand interactions. We have studied these issues in the N-terminal domain of protein L9 (NTL9), a model protein with mixed α/β structure. We have developed a general approach for introducing site-specific IR probes for the side chains (azide) and backbone (13C=18O) using recombinant protein expression. T-jump, time-resolved IR spectroscopy combined with site-specific labeling enables independent measurement of the respective backbone and side chain dynamics with single residue resolution. We find that side chain ordering in a key region of the β-sheet structure occurs on a slower time scale than ordering of the backbone during the folding of NTL9, likely due to the transient formation of nonnative side chain interactions.

Sequence, structure, and cooperativity in folding of elementary protein structural motifs

“…Muñoz and coworkers pioneered the multiprobe equilibrium approach for studies of "downhill" folding (5,7,8), where cooperativity is minimal, but subsequently demonstrated its applicability to other fast-folding proteins (9)(10)(11)(12)(13) and extended the analysis to kinetics (14). Following their work, experiments from other laboratories have reported probe-dependent folding equilibria and kinetics in a number of small proteins (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). However, because the spectroscopic signals do not directly report on structure and are often subject to interferences from nonstructural effects that lead to ambiguities in their interpretations, the challenge lies in relating the observed experimental data to the underlying structural and energetic states of the protein.…”

Mapping fast protein folding with multiple-site fluorescent probes

“…Noninvasive equilibrium NMR experiments, including some coupled with molecular dynamics simulations, have evaluated two-state fits and provided high resolution folding landscapes by using multiple probe nuclei (10)(11)(12). Notable progress in multiprobe analysis of folding kinetics has been achieved by using nonnatural amino acid IR probes (13) in combination with T-jump relaxation spectroscopy (14,15). Comparisons between fast protein folding kinetics probed by fluorescence and/or IR spectroscopy identified heterogeneous folding of trpzip2 (16), λ repressor (17), and α 3 D (18).…”

Synthesis of Enantiomerically Enriched 1,2,3- Triazole-derivatized Homoalanines

“…Merck Silica gel 60 F 254 plates were used for TLC analysis of reaction mixtures; visualization was done by UV light or by 1% ninhydrin in EtOH followed by heating. Infrared spectra were performed on Perkin-Elmer FT-IR system, 1 H-and 13 ( …”

“…The combined organic layer was washed with brine (2 × 10 mL), dried (Na 2 (2;4)), 2.52-2.35 (2m, 2H, H-C(3)). 13 ')), 1.31 (m, 6H, H-C(3''); H-C(4''); H-C(5'')), 0.88 (t, 3H, 3 J = 6.4 Hz H-C(6'')). 13 …”

“…Those include triazole tethered ferrocenyl-amino acids [10] and both C-and N-glycosyl α-amino acids [11,12]. On the other hand, both azide-and alkyne-modified amino acids were incorporated into pseudonatural peptides to gain site-specific recombinant infrared probes [13,14]. These modified amino acid residues were also used as bioorthogonal chemical reporters [15].…”