P450cam突变体蛋白表达、纯化与结晶的改进及其四突变体（F87L/Y96F/L244A/V247A）晶体结构

在P450cam突变体蛋白纯化过程中使用280 nm/392 nm双波长比值检测蛋白质纯度, 采用β-巯基乙醇对蛋白质进行还原性保护,有效地缩短了纯化进程,纯化效率相应提高.以PEG 8000为沉淀剂经悬滴汽相扩散法筛选得到适合衍射的P450cam四突变体（F87L/Y96F/L244A/V247A）晶体,在Mar-Research面探测器系统上收集了0.22 nm分辨率的X射线衍射数据.采用同晶差值傅立叶法解析结构,最后的晶体学R因子和R_free分别为0.197和0.247,键长偏差为0.001 77 nm,键角偏差为1.96°.结构测定显示P450cam四突变体（F87L/Y96F/L244A/V247A）和P450cam野生型的整体构象无重大变化,突变后活性口袋变大而疏水性增加,这与突变设计预期目标一致.     

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.     

Role of subunit interactions in P450 oligomers in the loss of homotropic cooperativity in the cytochrome P450 3A4 mutant L211F/D214E/F304W

The contribution of conformational heterogeneity to cooperativity in cytochrome P450 3A4 was investigated using the mutant L211F/D214E/F304W. Initial spectral studies revealed a loss of cooperativity of the 1-pyrenebutanol (1-PB) induced spin shift (S(50)=5.4 microM, n=1.0) but retained cooperativity of alpha-naphthoflavone binding. Continuous variation (Job's titration) experiments showed the existence of two pools of enzyme with different 1-PB binding characteristics. Monitoring of 1-PB binding by fluorescence resonance energy transfer from the substrate to the heme confirmed that the high-affinity site (K(D)=0.3 microM) is retained in at least some fraction of the enzyme, although cooperativity is masked. Removal of apoprotein on a second column increased the high-spin content and restored cooperativity of 1-PB binding and of progesterone and testosterone 6beta-hydroxylation. The loss of cooperativity in the mutant is, therefore, mediated by the interaction of holo- and apo-P450 in mixed oligomers.     

Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein

The mutant F99S/M153T/V163A of the Green Fluorescent Protein (c3-GFP) has spectral characteristics similar to the wild-type GFP, but it is 42-fold more fluorescent in vivo. Here, we report the crystal structure and the refolding properties of c3-GFP and compare them with those of the less fluorescent wt-GFP and S65T mutant. The topology and the overall structure of c3-GFP is similar to the wild-type GFP. The three mutated residues, Ser99, Thr153, and Ala163, lie on the surface of the protein in three different beta-strands. The side chains of Ser99 and Thr153 are exposed to the solvent, whereas that of Ala163 points toward the interior of the protein. No significant deviation from the structure of the wild-type molecule is found around these positions, and there is not clear evidence of any distortion in the position of the chromophore or of the surrounding residues induced by the mutated amino acids. In vitro refolding experiments on urea-denatured c3-GFP reveal a renaturation behavior similar to that of the S65T molecule, with kinetic constants of the same order of magnitude. We conclude that the higher fluorescence activity of c3-GFP can be attributed neither to particular structural features nor to a faster folding process, as previously proposed.