Residue-Specific Kinetic Insights into the Transition State in Slow Polypeptide Topological Isomerization by NMR Exchange Spectroscopy

Abstract

The characterization of the transition state is a central issue in biophysical studies of protein folding. NMR is a multiprobe measurement technique that provides residuespecific information. Here, we used exchange spectroscopy to characterize the transition state of the two-state slow topological isomerization of a 27-residue lantibiotic peptide. The exchange kinetic rates varied on a per-residue basis, indicating the reduced kinetic cooperativity of the two-state exchange, as well as the previously observed reduced thermodynamic cooperativity. Furthermore, temperature-dependent measurements revealed large variations in the activation enthalpy and entropy terms among residues. Interestingly, we found a linear relationship between the logarithm of the equilibrium constants and that of the exchange rates. Because the data points are derived from amino acid residues in one polypeptide chain, we refer to the linear relationship as the residue-based linear free energy relationship (rbLFER). The rbLFER offers information about the transition state of the twostate exchange.

Deviation from the Residue-Based Linear Free Energy Relationship Reveals a Non-Native Structure in Protein Folding

“…Type N is referred to as the negative correlation between log k and log k', which leads to the blue least-square lines with the slope of 0 < r < 1 in the REFER plot (cf. equation 9 in the previous paper [6]). In extreme cases, when the distribution of data points in the log k vs. log k' plot has a flattened shape, the least square lines have the slope of 0 or 1.…”

“…NMR provides local information around nuclei at an atomic resolution. We used NMR to determine the residue-specific equilibrium constants and residue-specific kinetic rate constants of a polypeptide chain in a two-state dynamic exchange, and discovered a linear relationship in the double logarithmic plot of the two parameters [6]. This type of plot is called a REFER (rate equilibrium free energy relationship) plot, and the linear relationship in this plot is generally referred to as the Linear Free Energy Relationship (LFER).…”

“…Residue-based LFER was discovered in the NMR study of a 27-residue bioactive peptide, nukacin ISK-1 [6]. The two sets of 1 H− 15 N amide cross peaks in the 1 H− 15 N HSQC (heteronuclear single quantum coherence) spectrum indicated the existence of two states in solution, termed states A and B [13].…”

“…The peak intensity ratio of the two cross peaks of each residue in the HSQC spectrum provides an equilibrium constant, K. Unexpectedly, we found that the K values varied from one residue to another [14]. Therefore, we determined the exchange rates, k (forward) and k' (backward), using EXSY (exchange spectroscopy) [6].…”

“…The first category (including nukacin ISK-1) is the EXSY NMR analyses of slow exchanges between two sets of cross peaks (Fig. 1A and 1B) [6,18,19], and the second one is the relaxation dispersion (RD) NMR analyses of the binding of an IDP (intrinsically denatured polypeptide) to a target protein (Fig. 1C) [20,21].…”