Moy F J, Seddon A P, Campbell E B, Böhlen P, Powers R
Department of Structural Biology, Wyeth-Ayerst Research, Pearl River, NY 10965, USA.
J Biomol NMR. 1995 Nov;6(3):245-54. doi: 10.1007/BF00197806.
The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising of 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation > 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C alpha and C beta chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C alpha correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H alpha and H beta protons as well as 3JHNH alpha coupling constants, amide exchange and 13C alpha and 13C beta secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel beta-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as beta-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the X-ray structures of FGF-2, where the first 17-19 residues were ill defined.
基于一系列三维三共振异核核磁共振实验，给出了重组人碱性成纤维细胞生长因子（FGF - 2）的1H、15N、13CO和13C共振归属。FGF - 2是一种由154个残基组成、分子量为17.2 kDa的蛋白质。这些研究使用了在大肠杆菌中表达的、同位素掺入率>95%的均匀标记的15N - 和15N - /13C - 标记的FGF - 2。序列特异性主链归属主要基于CBCA(CO)NH和HBHA(CO)NH实验中Cα、Cβ和Hα与下一个残基的主链酰胺1H和15N之间的残基间相关性，以及CBCANH和HNHA实验中Cα、Cβ和Hα与主链酰胺1H和15N之间的残基内相关性。此外，Cα和Cβ化学位移归属用于确定氨基酸类型。通过在HNCO和HCACO实验中观察到的羰基相关性以及HNCA实验中的Cα相关性来验证序列归属。脂肪族侧链自旋系统主要从H(CCO)NH和C(CO)NH实验中进行归属，这些实验将给定残基的所有脂肪族1H和13C共振与下一个残基的酰胺共振相关联。通过HCCH - COSY和HCCH - TOCSY实验进行额外的侧链归属。FGF - 2的二级结构基于涉及NH、Hα和Hβ质子的NOE数据以及3JHNHα耦合常数、酰胺交换和13Cα和13Cβ二级化学位移。结果表明，FGF - 2由11个明确的反平行β - 折叠（残基30 - 34、39 - 44、48 - 53、62 - 67、71 - 76、81 - 85、91 - 94、103 - 108、113 - 118、123 - 125和148 - 152）和一个螺旋状结构（残基131 - 136）组成，它们主要通过紧密转角相连。该结构与FGF - 2的精制X射线晶体结构不同，在X射线晶体结构中，残基131 - 136被定义为β - 链XI。在主要肝素结合位点（残基128 - 138）发现螺旋状区域而非X射线结构中描述的β - 链构象，可能对理解肝素 - FGF - 2相互作用的本质具有重要意义。此外，N末端残基9 - 28在溶液中存在两种不同的构象。这与FGF - 2的X射线结构一致，其中前17 - 19个残基定义不明确。
