Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.

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REF/Yra1p interacts with TAP/Mex67p, and in yeast this interaction leads to displacement of Sub2p from Yra1p (7). TAP heterodimerizes with p15 and binds nucleoporins through central and C-terminal domains (8), directing the mRNP to the nuclear pore and promoting transport to the cytoplasm. On the cytoplasmic side of the nuclear pore, Dbp5p triggers displacement of Mex67p from mRNA. Yra1p binds mRNA early during its nuclear maturation but is no longer bound once it reaches the nuclear periphery (9). Consistent with this finding, analysis of Balbiani ring pre-mRNPs shows that UAP56 and REF accompany the mRNP to the nuclear periphery where UAP56 and then REF dissociate during translocation through the pore (10).Although Yra1p is essential for yeast mRNA export, depletion of REF in higher eukaryotes does not block bulk mRNA export (11), suggesting that other proteins can fulfill this role and that there may be functional redundancy between export adaptors. The shuttling SR proteins 9G8, SRp20, and SF2/ASF directly bind TAP by short arginine-rich peptides (12, 13) and can function as export factors (14). Even in yeast, other proteins can recruit Mex67p to the mRNP, including Yra2p (15) and Npl3p.The fact that TAP binds RNA weakly in vitro led to the idea that export factors such as REF, which bind RNA avidly, bridge the interaction between TAP and mRNA, leading to the term mRNA export adaptors (15). However, both TAP and Mex67p are readily UV-cross-linked to mRNA in vivo (16)(17)(18), suggesting a direct stable interaction at some point during export. Here, we show that mRNA is handed over from export adaptors to TAP and that at least in vitro, export adaptors have the ability to enhance the RNA-binding activity of TAP. ResultsThe RNA-and TAP-Binding Sites on REF Overlap. In Saccharomyces cerevisiae, Mex67p binding to Yra1p triggers displacement of Sub2p (7), so we established whether this is the case for the mammalian orthologs. We examined whether UAP56 coimmunoprecipitated (Co-IP) with REF2-I (REF) in the presence of increasing amounts of TAP. This analysis revealed that TAP triggered dissociation of UAP56 from REF (Fig. 1A, lanes 5 and 6).We analyzed organization of the resulting REF-TAP-RNA ternary complex by examining how REF binds RNA. NMR

The sequence-specific RNA-binding proteins SRp20 and 9G8 are the smallest members of the serine-and arginine-rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5 0 CAUC3 0 . The SRp20-RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5 0 cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine-rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip-associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.

The cytochrome P450 responsible for the debrisoquine/sparteine polymorphism (P450 2D6) has been produced in large quantities by expression of a modified cDNA in baculovirus. A polyhistidine extension was incorporated at the C-terminus of the expressed protein, which, after purification of the protein on a nickel-agarose column, could be removed proteolytically by treatment with thrombin. Purified yields of P450 2D6 were 2.4 mg from 700 mL of cell culture. The protein had a greater than 90% heme content and was fully active, having no residual absorbance at 420 nm in the reduced CO complex. The quantities produced allowed direct study of the interaction of the substrate codeine with the enzyme by paramagnetic relaxation effects on the NMR spectrum of the substrate. Distances between the heme iron atom and substrate protons were calculated from these experiments, and the orientation of the substrate in the binding pocket was determined. This showed that codeine was bound with the methoxy group of the molecule closest to the heme iron (iron-methyl proton distance of 3.1 +/- 0.1 A), consistent with the observed O-demethylation to morphine. A model of the complex Of P450 2D6 with codeine was built from a multiple sequence and structure alignment of the known crystal structures for P450s, incorporating the experimental constraints derived from the NMR studies. This showed that the overall fold Of P450 2D6 is more similar to that of P450 BM3 than to either P450 cam or P450 terp. Codeine binds to P450 2D6 so that the methoxy group is directly above the A ring of the heme, while the basic nitrogen interacts with the carboxylate of aspartate 301.