Solid-state NMR yields structural constraints on the V3 loop from HIV-1 Gp120 bound to the 447-52D antibody Fv fragment
Solid-state NMR yields structural constraints on the V3 loop from HIV-1 Gp120 bound to the 447-52D antibody Fv fragment
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DOI：
10.1021/ja0392162
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发表时间：
2004-04-21
影响因子：
15
通讯作者：
Tycko, R
中科院分区：
文献类型：
--
作者：
Sharpe, S;Kessler, N;Tycko, R
在冷冻的甘油/水溶液中，对来自HIV-1 MN株的gp 120包膜糖蛋白的V3环序列的18-残基肽与人抗gp 120单克隆抗体447- 52 D的Fv片段的复合物进行固态NMR测量。该肽在含有表位中保守的GPGR基序的7个残基片段中被均匀地N-15-和C-13-标记。从二维C-13-C-13和N-15-C-13魔角旋转NMR谱中获得标记片段的N-15和C-13 NMR化学位移归属。C-13 NMR谱线宽度的减少和复合物形成后化学位移的变化表明采用了明确定义的抗体依赖性结构。分子内C-13-C-13的复合物中的距离，这限制了在GPGR基序的肽骨架和侧链构象，从旋转共振（RR）数据的分析确定。从化学位移和RR测量的结构约束是在最近的解决方案NMR和结晶学研究这个系统的良好协议，虽然注意到某些肽侧链的结构排序的差异。这些实验探索和帮助描绘的实用程序的固态NMR技术作为结构探针的肽/蛋白质复合物一般，可能包括膜相关的激素/受体复合物。
Solid-state NMR measurements were performed on the complex of an 18-residue peptide derived from the V3 loop sequence of the gp120 envelope glycoprotein of the HIV-1 MN strain with Fv fragments of the human anti-gp120 monoclonal antibody 447-52D in a frozen glycerol/water solution. The peptide was uniformly N-15- and C-13-labeled in a 7-residue segment containing the conserved GPGR motif in the epitope. N-15 and C-13 NMR chemical shift assignments for the labeled segment were obtained from two-dimensional C-13-C-13 and N-15-C-13 magic-angle spinning NMR spectra. Reductions in C-13 NMR line widths and changes in chemical shifts upon complex formation indicate the adoption of a well-defined, antibody-dependent structure. Intramolecular C-13-C-13 distances in the complex, which constrain the peptide backbone and side chain conformations in the GPGR motif, were determined from an analysis of rotational resonance (RR) data. Structural constraints from chemical shifts and RR measurements are in good agreement with recent solution NMR and crystallographic studies of this system, although differences regarding structural ordering of certain peptide side chains are noted. These experiments explore and help delineate the utility of solid state NMR techniques as structural probes of peptide/protein complexes in general, potentially including membrane-associated hormone/receptor complexes.