4.3. Conformational Analysis

NMR Spectroscopy. Samples were prepared by dissolving peptides in 0.54 mL of H₂O and 0.06 mL of D₂O (pH 5.5), to obtain a 1 mM final concentration. NMR spectra were recorded on an INOVA 700 MHz spectrometer (Varian, Palo Alto, CA, USA) equipped with a z-gradient 5 mm triple-resonance probe head at 25 °C. One-dimensional (1D) NMR spectra were recorded in the Fourier mode with quadrature detection. The water signal was suppressed by gradient echo [44]; 2D DQF-COSY [45,46], TOCSY, [47] and NOESY [48] spectra were recorded in the phase-sensitive mode using the method from States et al. [49]. Data block sizes were 2048 addresses in t2 and 512 equidistant t1 values. Before Fourier transformation, the time domain data matrices were multiplied by shifted sin² functions in both dimensions. A mixing time of 80 ms was used for the TOCSY experiments. NOESY experiments were run with a 100 ms mixing time. The qualitative and quantitative analyses of DQF-COSY, TOCSY, and NOESY spectra, were obtained using the interactive program package XEASY [50]. Almost complete ¹H-NMR chemical shift assignments were effectively achieved for Pep 1 and for the main conformer of Pep 2 according to the Wüthrich [51] procedure via the usual systematic application of DQF-COSY, TOCSY, and NOESY experiments with the support of the XEASY software package (Tables S1 and S2). The NOE-based distance restraints were obtained from NOESY spectra of Pep 1 (Table S3). The NOESY cross peaks were integrated with the XEASY program and were converted into upper distance bounds using the CALIBA program incorporated into the program package CYANA [52]. Only NOE derived constraints were considered in the annealing procedures. An ensemble of 100 structures was generated with the simulated annealing of the program CYANA. Then, 10 structures were chosen, whose interproton distances best fitted NOE derived distances, and refined through successive steps of restrained and unrestrained energy minimization calculations using the Discover algorithm (Accelrys, San Diego, CA, USA) and the consistent valence force field [53]. The minimization lowered the total energy of the structures; no residue was found in the disallowed region of the Ramachandran plot. The final structures were analyzed using the InsightII program (Accelrys). Molecular graphics images were realized using the UCSF Chimera package [54].