Unfolding and refolding pathways of numerous proteins have been studied using a variety of different redox systems and solution conditions (Jaenicke and Rudolph 1989). The protein structural transitions that occur in the pathway are at the heart of the unfolding and refolding processes. Dynamic analysis of the unfolding/refolding pathways and identification of the specific conformational changes that form the individual intermediates involved in the rate-limited pathway(s) can distinguish one pathway from another (Rothwarf and Scheraga 1993a, b). Such studies in turn provide new insights into the functional properties and mechanisms of proteins that will lead to a more detailed and more complete description of biological functions (Alexandrescu et al. 1998). It is usually of considerable interest to estimate the conformational changes both of the whole protein tertiary structure in solution and of specific sites observed by spectroscopic techniques. We propose extending these investigations by image analysis through the use of 1H nuclear magnetic resonance (NMR) spectroscopy, which plays a prominent role in such studies because of its ability to follow protein unfolding and refolding with a direct, continuous, nondestructive method at an atomic level to yield detailed information about the structure of small proteins in solution (for review, see Wagner 1997).
Bovine pancreatic ribonuclease A (RNase A), which is well characterized as a single-domain protein that contains 124 residues with four native disulfide bonds, has played a crucial role as a model system in studies of protein structure, folding, and enzyme catalysis. Its native state has been studied by using NMR (Rico et al. 1989; Santoro et al. 1993). The refolding and unfolding pathway of RNase A has been studied extensively by using NMR and other biophysical techniques (for review, see Neira and Rico 1997). Later studies suggested that RNase A folds and unfolds through multiple pathways determined by several rate-limited transition intermediates (Rothwarf and Scheraga 1993b; Li et al. 1995). The complexity of the multiple pathways means that different mechanisms may occur with different types of redox systems and different solution conditions (Rothwarf and Scheraga 1993c). A comprehensive understanding of the folding and unfolding process of RNase A requires that the different conditions and methods for fundamental experiments must be stated carefully.
NMR studies of RNase A have led to assignments for the spin systems of the 124 amino acids residues and sequence-specific assignments by using 1H NMR, 2-D 1H-1H homonuclear correlation spectra and amide proton/deuterium exchange (Rico et al. 1989; Robertson et al. 1989). These assignments were used to locate conformational changes associated with the binding of nucleotides, substrates, and inhibitors to RNase A and with chemical modifications of RNase A. Recent advances in NMR spectroscopy, primarily through the use of triple-resonance experiments involving uniformly 15N- and 13C-enriched proteins, have provided new insights into the structure, stability, and conformational dynamics of proteins (Cavanagh et al. 1995). NMR methods have been used to characterize the solution structure of the major folding intermediates of proteins and analogs of the rate-determining intermediates (Adler and Scheraga 1990; Talluri et al. 1994; Laity et al. 1997; Shimotakahara et al. 1997). NMR spectroscopy also has been used to monitor indirectly the kinetics of regular backbone structural information in RNase A during unfolding and refolding (Biringer and Fink 1982; Zhang et al. 1995; Alexandrescu et al. 1998). Although NMR can provide extensive conformation and structural information by chemical shift (Wishart et al. 1991), scalar coupling constants (Smith et al. 1996), NMR peak intensities (Biringer and Fink 1982), nuclear Overhauser enhancement (NOE) (Williams 1989), resonance linewidth in a NMR spectrum, and other specific structural restraints that represent a time average over the ensemble of conformational substates, few dynamic NMR measurements have been recorded for relatively large native proteins such as RNase A because of the limits imposed by significant overlap of the resonances in the 1H NMR spectra and the excessively long times needed for homonuclear 2-D NMR spectra to record the rapid changes in the dynamic process. To overcome these limits, investigators have used different methods such as hydrogen exchange (Zhang et al. 1995) and 15N backbone labeling (Alexandrescu et al. 1998) in NMR studies of the dynamics of RNase A.
In traditional NMR studies, some specific nuclear resonances have to be assigned to conduct protein dynamics investigations. In this article, the whole spectrum in H2O for image analysis was obtained, and no assignments were made as usual. We propose to analyze the complex 1-D NMR data by using image analysis algorithms. Three spectral parameters including Shannon entropy, mutual information, and correlation coefficient were introduced into the analysis. The kinetic patterns then were characterized by these parameters. The primary goal of the present article is to introduce a new insight into the dynamic investigation of the unfolding/refolding pathway of RNase A in reduced dithiothreitol (DTT). The specific objectives are: (1) to determine conditions that are slow enough for 1H NMR (and for 2-D NMR) monitoring; (2) to calculate the image specific parameters of the unfolding pathways; and (3) to compare the denatured pathways at different temperatures. The present study used image analysis of the 1H NMR spectra to focus on the detailed kinetic data analysis of all of the conformational changes occurring when a protein unfolds.