In the past, BSA crystals have been grown by a vapor diffusion technique from 50 mM potassium phosphate buffer, pH 6.2, 52% saturated ammonium sulphate at 298 K [15–17]. However, these crystals, which belong to space group P6 with unit cell parameters a = b =148.24 Å, c = 356.70 Å and α = 90°, β = 90°, γ = 120°, only diffract at low resolution (8–10 Å) [15,16].

Screening using polyethylene glycol of different molecular weights (2000–20,000 Da) as precipitating agent revealed new conditions for the crystallization of BSA. In particular, thin, small and fragile crystals appeared within 7 days using 30 mg mL⁻¹ protein concentration with the hanging-drop method from crystallization conditions in which the reservoir solution contained 24% w/v MPEG 2K, 0.1 M Tris HCl pH 8. The quality of the crystals was improved by fine-tuning the concentration of protein (10.0–60.0 mg mL⁻¹), changing the precipitants and their concentration, and evaluating the effect of divalent cations, such as CaCl₂, ZnCl₂, MgCl₂. The best crystals (Figure 1a–e) were obtained from a crystallization solution containing 22–24% w/v MPEG 5K, 0.2M MgCl₂, 0.1 M Tris HCl pH 7.8, 8.0 and 8.2 and BSA at 20.0 mg mL⁻¹. Further optimizations of the crystallization conditions to grow larger and thicker crystals suitable for diffraction data collection at high resolution, using other methods (sitting drops or microbatch without oil [20]) failed.

Various cryosolutions (20% v/v glycerol, 300 mg mL⁻¹ trehalose, 300 mg mL⁻¹ saccharose) were prepared to examine their ability to cryoprotect the BSA crystals. Preliminary X-ray diffraction data collected at 100 K showed that even the best crystals (Figure 1a,b) were intrinsically disordered and that the largest ones diffracted at most to 8 Å resolution using glycerol as cryoprotectant. Application of an annealing protocol failed to improve the crystal diffraction quality. The latter method transiently returns the flash-cooled crystal to ambient temperature and has been shown to improve poor resolution and mosaicity, presumably caused by incorrect flash-cooling [21,22]. However, as reported in other cases [18,19,23–26], we found an increase in the diffraction power of BSA crystals by dehydration. A number of different trials for dehydrating crystals have been described in the literature. A comprehensive survey of the successfully used dehydration procedures is reported in Table 1 [18,19,24–85]. The dehydration process has been applied with success to crystals of proteins of various molecular weights, protein-protein and protein-ligand complexes. The resolution of the diffraction data collected from dehydrated crystals ranges from 1.1 Å to 4.5–5 Å, with resolution improvements that in some cases have been >10 Å; while the solvent content values range from 23% to 85%, with a decrease upon dehydration that generally has been <10%. The values of relative humidity in equilibrium with the solutions of the examined systems range from 74.3% to 99.5%. As expected, the best improvements in the X-ray diffraction power of protein crystals have been observed when the dehydration process has been applied to crystals with the highest solvent contents. Notably, the analysis of the Table suggests that even small changes in solvent content and relative humidity can promote favorable lattice rearrangements that dramatically improve the diffraction properties of crystals, as recently suggested by Russi et al. [26]. These findings underline the importance of reproducible and controlled crystal dehydration, such as that which can be obtained using modern devices available at synchrotron beamlines [86–88]. The data also confirm that at the start of a dehydration experiment, the relative humidity in equilibrium with the mother liquor is very often close to 100%, in agreement with recent data [89].

Various dehydration protocols have been used. The dehydration process traditionally consists of equilibrating the protein crystals over a reservoir with a higher percentage of precipitant [24,28–35]. The hanging drop containing the crystals is then allowed to dehydrate for 12 h to 3 days. The simplest implementation involves dehydration by air [25,36–42]. Good results have been also obtained when protein crystals are mounted in a specific and adjustable stream of humidified gas, where it is possible to control the relative humidity [26,43–48,86–88]. Finally, crystal dehydration can also be performed by transferring the crystals into a dehydrating solution, which is the original mother liquor with a higher concentration of precipitant [24,27,50–70] or with a different dehydrating agent [49,71–85].

In the present case, common cryoprotectants, various salts (for example malonate) and different molecular-weight PEGs were tested as possible dehydration agents, but ultimately the most successful experiment was obtained when crystals which were grown in 22–24% w/v MPEG 5K, 0.2 M MgCl₂, 0.1 M Tris HCl pH 7.8 were directly transferred to a solution containing 30% w/v PEG 8K, 0.1M MgCl₂, 0.05 M Tris HCl pH 7.8. Crystals did not show any signs of cracking during dehydration. After dehydration and cryocooling, the diffraction resolution of the crystals on the in-house X-ray equipment improved to 3.24 Å resolution. The diffraction resolution could be even further improved with a synchrotron radiation source. Assuming the presence of two BSA molecules in the asymmetric unit, the crystal volume per unit molecular weight (V_M) is 2.3 ų Da⁻¹, with a solvent content of 47%, which is within the normal range for protein crystals [92]. The solvent content of the crystals was reduced by 3–6% by dehydration. This process also produces a change in their relative humidity from 99.2% to 98.5%.

The application of molecular replacement, as detailed in the Experimental Section, enabled the identification of orientation and position of the two molecules in the asymmetric unit that gave a satisfactory fit to the experimental data. Refinement of the model, obtained by molecular replacement using phases derived from the structure of HSA is in progress.

The structural determination will provide a molecular basis for explaining numerous physical phenomena and for future docking and molecular dynamics studies on BSA complexes with drugs and other bioactive small molecules.

Bovine serum albumin fraction V and all other reagents were purchased from Sigma Chemical Co. and used as supplied without further purification. BSA (80 mg/mL) was dissolved in 10 mM Tris-HCl buffer, pH 7.8. The protein concentration was determined spectrophotometrically using the extinction coefficient of 36,500 M⁻¹ cm⁻¹ at 280 nm [93].

Crystallization trials were performed at 293 K by the hanging-drop or sitting drop vapor-diffusion methods with 0.5 μL of protein and 0.5 μL of precipitant solution and a reservoir volume of 500 μL or using the microbatch without oil method [20] with the same volumes. Initial screens have included systematic PEG/pH and PEG/Ion screens. In particular, we prepared solutions with a formulation similar to the commercially available kits of Hampton Research. More than 100 different conditions were examined. In these crystallization experiments we varied the concentration of PEG from 10% w/v to 30% w/v, the molecular weight of PEG from 2000 Da to 20,000 Da and the pH from 7 to 8. The effect of divalent cations, such as CaCl₂, ZnCl₂, MgCl₂ was also evaluated.

Needle crystals were obtained within 7 days from drops containing BSA (30 mg mL⁻¹ in 10 mM Tris-HCl, pH 7.4) 24% w/v MPEG 2K and 0.1 M Tris HCl pH 8. An improvement in the quality of crystals was obtained using different salts and precipitant agents. In particular, well shaped crystals were grown using 22% w/v MPEG 5K, 0.2 M MgCl₂, 0.1 M Tris HCl pH 7.8 as a precipitant solution. These crystals diffracted to 8 Å resolution. In all the experiments, standard 24-well linbro plates (Hampton Research, Laguna Niguel, USA) were used.

A significant improvement in the crystal diffraction quality was obtained by dehydration with PEG 8K. In this procedure, protein crystals were transferred in a loop to a 5 μL solution containing 30% w/v PEG 8K, 0.05 M Tris HCl pH 7.8 and 0.1 M MgCl₂ for 10 min in the open air. After dehydration, the crystals were cryoprotected by soaking for 5–10 s in a solution consisting of 30% w/v PEG 8K, 0.05 M Tris HCl pH 7.8 and 0.1 M MgCl₂, 20% v/v glycerol and tested for diffraction quality as above.

X-ray diffraction data (3.24 Å resolution) were collected at the Institute of Biostructures and Bioimages (Naples, Italy), at 100 K using a Rigaku MicroMax-007 HF generator producing Cu Kα radiation and equipped with a Saturn944 CCD detector. An oscillation range of 0.5° and an exposure time of 55 s were adopted for the experiments. The data sets were indexed, processed and scaled using the HKL-2000 package (Table 2) [94].

The overall R_merge was high at 15.4% and the R_merge value in the highest resolution bin was 31.9%. We attribute the high R_merge value as being primarily due to the large number of weak reflections that were measured and maybe to some radiation damage.

The structure of the protein was solved by molecular replacement using the program Phaser [95] and HSA as search model (PDB code 2AO6 [96]). Water molecules were removed from the model prior to structure factor and phase calculations. The solution had an R-factor of 0.39.