The dynamic properties of RK peptide both free and coupled to a model carrier protein (BPTI) were determined using multidimensional, natural abundance 13C NMR techniques. This peptide is derived from the third hypervariable region of the gp120 protein envelope of HIV-1, isolate BH-10, and has the sequence RIQRGPGRAFVTIGK. This region represents the principle neutralization domain (PND) of HIV-1, and corresponds to residues 308-322 of the gp120 protein envelope. A C-terminal, cysteinyl version of RK peptide, RKC, was successfully conjugated to the carrier protein using established procedures (Ebina et al., 1989). Relaxation experiments (R(Cz), R(C xy), and NOE) were carried out on the peptide in each state. The relaxation rate data was used to calculate an approximate spectral density function (SDF) for each residue in the peptide. Two SDF approximations in particular were investigated: the reduced and high frequency (HF) approximations. A molecular dynamics investigation of each approximation was performed based on a model protein system, staphylococcal nuclease (SNase) in order to obtain a realistic functional form for the SDF. In addition, experimental full SDF mappings were carried out on a 13Cα labeled version of RKC peptide (RKC313), native BPTI, and a 13Cα labeled alanine sample. In this way, the applicability of each approximation to small, disordered systems was investigated theoretically and experimentally. It was determined that the reduced approximation is more amenable to small, disordered systems (e.g. RKC peptide), while the HF approximation was more suitable for larger, well-ordered molecules in solution (e.g. native BPTI). We have determined that for very large or very small molecules there exists domains of order that are more amenable to each approximation. In general, lower order is more amenable to the reduced approximation. In many cases, the choice of approximation becomes critical, as the difference in error between each approximation is greater than the expected experimental error.
Using these approximations, it was determined that free RKC is largely unstructured in solution, consistent with previous structural studies on this molecule. Upon covalent attachment of RKC to BPTI, there was increased restriction of each residue upon approaching the binding site. In addition, there was increased restriction of the Arg8 bond vector. This observation supports previous structural studies of this molecule for which NOEs consistent with transient formation (<10%) of a type I or type II β-turn in the GPGR region were observed.
|School:||University of Cincinnati|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Keywords:||Bpti, Relative errors, Rkc, Sdf|
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