L-Proline is one of Mother Nature's cryoprotectants. Plants and yeast accumulate proline under freeze-induced stress and the use of proline in the cryopreservation of biological samples is well established. Here, it is shown that L-proline is also a useful cryoprotectant for protein crystallography. Proline was used to prepare crystals of lysozyme, xylose isomerase, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase for low-temperature data collection. The crystallization solutions in these test cases included the commonly used precipitants ammonium sulfate, sodium chloride and polyethylene glycol and spanned the pH range 4.6-8.5. Thus, proline is compatible with typical protein-crystallization formulations. The proline concentration needed for cryoprotection of these crystals is in the range 2.0-3.0 M. Complete data sets were collected from the proline-protected crystals. Proline performed as well as traditional cryoprotectants based on the diffraction resolution and data-quality statistics. The structures were refined to assess the binding of proline to these proteins. As observed with traditional cryoprotectants such as glycerol and ethylene glycol, the electron-density maps clearly showed the presence of proline molecules bound to the protein. In two cases, histidine acid phosphatase and 1-pyrroline-5-carboxylate dehydrogenase, proline binds in the active site. It is concluded that L-proline is an effective cryoprotectant for protein crystallography.

The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate) by NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in complex with NADH and of the oxidized Glu28Gln enzyme in complex with CH 3 -H 4 folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005) Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile active site residue. In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex in order to stack against the p-aminobenzoate ring of the folate. Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not invariant; for example, a leucine occurs at this site in the human enzyme. To examine the role of Phe223 in substrate binding and catalysis, we have constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate that Phe223 participates in the binding of both substrates. The Phe223Ala mutation impairs NADH and CH 2 -H 4 folate binding each 40-fold, yet slows catalysis of both half-reactions less than 2-fold. Affinity for CH 2 -H 4 folate is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative half-reaction 3-fold faster than the wild-type enzyme. Structures of ligandfree Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH 3 -H 4 folate have been determined at 1.65 Å and 1.70 Å resolution, respectively. The structures show that the folate is bound in a catalytically competent conformation, and Leu223 undergoes a conformational change similar to that observed for Phe223 in the Glu28Gln-CH 3 -H 4 folate structure. Taken together, our results suggest that Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of E. coli MTHFR. † This work was supported in part by the American Chemical Society Petroleum Research Fund Grant 39599-GB4 (E.E. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 18. Published in final edited form as:Biochemistry. Methylenetetrahydrofolate reductase (MTHFR) 1 is a flavoprotein that catalyzes the NAD(P) H-dependent reduction of 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate), as shown in eq 1.(1)The reaction provides CH 3 -H 4 folate, which is the sole methyl donor to homocysteine in the production of methionine by methionine synthase. MTHFR plays a significant role in the homeostasis of homocysteine; mutations in the enzyme lead to hyperhomocyst(e)inemia, (1, 2), which is associated with an increased risk for the development of cardiovascular disease (reviewed in (3)) and Alzheimer's disease (4-6) in adults, and of neural tube defects in the fetus (reviewed in (7)).MTHFRs from pig liver, human, E. coli, yeast, Arabidopsis, and Leishmania have been characterized (reviewed in (8); (9-12)). Whereas the mammalian and yeast MTHFRs are h...