Santos Sandra P, Bandeiras Tiago M, Pinto Ana F, Teixeira Miguel, Carrondo Maria A, Romão Célia V
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Protein Expr Purif. 2012 Feb;81(2):193-200. doi: 10.1016/j.pep.2011.10.001. Epub 2011 Oct 24.
Desulforubrerythrin from Campylobacter jejuni has recently been biochemical and spectroscopically characterized. It is a member of the rubrerythrin family, and it is composed of three structural domains: the N-terminal desulforedoxin domain with a non-heme iron center, followed by a four-helix bundle domain harboring a binuclear iron center and finally a C-terminal rubredoxin domain. To date, this is the first example of a protein presenting this kind of structural domain organization, and therefore the determination of its crystal structure may unveil unexpected structural features. Several attempts were made in order to obtain protein crystals, but always without success. As part of our strategy the thermofluor method was used to increase protein stability and its propensity to crystallize. This approach has been recently used to optimize protein buffer formulation, thus yielding more stable and homogenous protein samples. Thermofluor has also been used to identify cofactors/ligands or small molecules that may help stabilize native protein states. A successful thermofluor approach was used to select a pH buffer condition that allowed the crystallization of Campylobacter jejuni desulforubrerythrin, by screening both buffer pH and salt concentration. A buffer formulation was obtained which increased the protein melting temperature by 7°C relatively to the initial purification buffer. Desulforubrerythrin was seen to be stabilized by lower pH and high salt concentration, and was dialyzed into the new selected buffer, 100mM MES pH 6.2, 500mM NaCl. This stability study was complemented with a second thermofluor assay in which different additives were screened. A crystallization screening was carried out and protein crystals were rapidly obtained in one condition. Protein crystal optimization was done using the same additive screening. Interestingly, a correlation between the stability studies and crystallization experiments using the additive screening could be established. The work presented here shows an elegant example where thermofluor was shown to be a key biophysical method that allowed the identification of an improved buffer formulation and the applicability of this technique to increase the propensity of a protein to crystallize is discussed.
空肠弯曲菌的脱硫红藻红素最近已通过生化和光谱学方法进行了表征。它是红藻红素家族的一员，由三个结构域组成：N端具有非血红素铁中心的脱硫铁氧还蛋白结构域，接着是含有双核铁中心的四螺旋束结构域，最后是C端的红素氧还蛋白结构域。迄今为止，这是呈现这种结构域组织的蛋白质的首个实例，因此其晶体结构的测定可能会揭示出意想不到的结构特征。为了获得蛋白质晶体进行了多次尝试，但均未成功。作为我们策略的一部分，热荧光法被用于提高蛋白质稳定性及其结晶倾向。这种方法最近已被用于优化蛋白质缓冲液配方，从而得到更稳定、更均一的蛋白质样品。热荧光法也已被用于鉴定可能有助于稳定天然蛋白质状态的辅因子/配体或小分子。通过同时筛选缓冲液pH值和盐浓度，采用成功的热荧光法选择了一种pH缓冲条件，该条件允许空肠弯曲菌脱硫红藻红素结晶。获得了一种缓冲液配方，相对于初始纯化缓冲液，该配方使蛋白质的解链温度提高了7°C。脱硫红藻红素在较低pH值和高盐浓度下被观察到是稳定的，并被透析到新选择的缓冲液中：100mM MES pH 6.2，500mM NaCl。这项稳定性研究通过第二项热荧光分析得到补充，在该分析中筛选了不同的添加剂。进行了结晶筛选，并在一种条件下迅速获得了蛋白质晶体。使用相同的添加剂筛选进行了蛋白质晶体优化。有趣的是，可以在稳定性研究与使用添加剂筛选的结晶实验之间建立相关性。此处展示的工作是一个很好的例子，其中热荧光法被证明是一种关键的生物物理方法，它允许鉴定出改进的缓冲液配方，并讨论了该技术在提高蛋白质结晶倾向方面的适用性。
