Two-dimensional NMR experiments have been performed on a peptide, succinyl-AE-TAAAKFLRAHA-NH2, related to the amino-terminal sequence of ribonuclease A. This peptide contains 50-60% helix in 0.1 M NaCl solution, pH 5.2, 3 degrees C, as measured by circular dichroism. NOESY spectra of the peptide in aqueous solution at low temperatures show a number of NOE connectivities that are used to determine the highly populated conformations of the peptide in solution. Short-range dNN(i, i + 1) and d alpha N(i, i + 1) connectivities and medium-range d alpha beta(i, i + 3) and d alpha N(i, i + 3) connectivities are detected. The pattern of NOE connectivities unambiguously establishes the presence of helix in this peptide. The magnitudes of the 3JHN alpha coupling constants and the intensities of the dNN(i, i + 1) and d alpha N(i,i + 1) NOEs allow the evaluation of the position of the helix along the peptide backbone. These data indicate that the amino terminus of the peptide is less helical than the remainder of the peptide. The observation of several long-range NOEs that are atypical of helices indicates the presence of a high population of peptide molecules in which the first three residues are distorted out of the helical conformation. The absence of these NOEs in a related peptide, RN-31, in which Arg 10 has been changed to Ala, suggests that this distortion at the amino-terminal end of the peptide arises from the formation of a salt bridge between Glu 2 and Arg 10.(ABSTRACT TRUNCATED AT 250 WORDS)
After the recent discovery of a ribonuclease A unfolding intermediate Nature 375, 513-515], we investigated the unfolding pathway of hen egg white lysozyme. At pH* 4.00 with D 2 O at 10°C and 6 M guanidinium chloride, unfolding shows a single, slow kinetic phase, with a relaxation time of 3300 s when monitored by circular dichroism (CD). Exchange of the tryptophan indole nitrogen protons shows that buried Trp residues 123, 111, and 108 lose tight packing and become solvent-exposed simultaneously, with a mean relaxation time of 3300 s, similar to the CDmonitored unfolding rate. Unfolding monitored by Trp fluorescence shows, moreover, that 90% of the amplitude change occurs in a slow phase, with a relaxation time of 2400 s. Faster-unfolding phases with minor amplitudes are detected by Trp indole hydrogen exchange and by fluorescence. It is likely that these changes are caused by Trp 62 and Trp 63, active site residues which are not buried in the hydrophobic core. Lysozyme unfolding was further monitored by the histidine 15 C 1 proton, which gives resolved lines for the native and unfolded species in one-dimensional 1 H-NMR spectra. The majority of the unfolding reaction, 70%, occurs in a slow phase with a relaxation time of 3600 s, but there is also a rapid unfolding phase; 30% of the His 15 C 1 proton resonance intensity is found at the unfolded chemical shift within tens of seconds after the start of unfolding. The amplitude of the rapid unfolding phase increases proportionally with the concentration of GdmCl denaturant present. These results show that a partially buried residue of lysozyme, histidine 15, takes part in forming an unfolding intermediate similar A. 92, 9318-9322]; the difference between that study and ours may reside in the greater sensitivity of 19 F to the detection of motional differences.Kinetic studies of the unfolding reactions of globular proteins monitored by CD 1 or fluorescence show that proteins typically unfold in a single kinetic step, N f U, with no detectable intermediates (Schmid, 1992; Mücke & Schmid, 1994). Thus, it was surprising when Kiefhaber et al. (1995) These results are startling. To test the generality of this behavior, we decided to study the unfolding of hen egg white lysozyme. Hen egg white lysozyme is composed of two domains: an R-domain that contains several helices and a -domain comprised largely of -strands (Figure 1). Lysozyme has many desirable characteristics for this work; its NMR assignments are known (Redfield & Dobson, 1988), and its refolding behavior has been studied in depth (Kiefhaber, 1995;Itzhaki et al., 1994;Dobson et al., 1994). Lysozyme unfolds very slowly, making the study of unfolding by 1D 1 H-NMR practical. Five of the protein's six tryptophan indole nitrogen protons give unique lines in 1D 1 H-NMR spectra. Upon denaturation, these lines disappear and the indole protons resonate in a common line at 10.06 ppm. Moreover, when the Trp side chain is exposed to D 2 O, the indole proton is exchanged for a deuteron and the NMR line disapp...
Perchlorate-denatured ribonuclease A (PDR) is known to show a circular dichroism (CD) spectrum suggestive of substantial secondary structure. Thus, PDR may be a molten globule form of ribonuclease A. We find that any secondary structure present in PDR does not provide measurable protection against amide proton exchange, and PDR does not belong to the class of structured molten globules. CD spectra of short peptides show that the perchlorate anion affects these spectra in a way that could be mistaken for secondary structure formation; thus, caution must be used in interpreting CD spectra of peptides and proteins taken in perchlorate solutions.
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