The X‐ray crystallographic structures of two mutants (K206Q and H207E) of the N‐lobe of human transferrin (hTF/2N) have been determined to high resolution (1.8 and 2.0 Å, respectively). Both mutant proteins bind iron with greater affinity than native hTF/2N. The structures of the K206Q and H207E mutants show interactions (both H‐bonding and electrostatic) that stabilize the interaction of Lys296 in the closed conformation, thereby stabilizing the iron bound forms.

The N-lobe of human serum transferrin (hTF/2N) has been expressed in baby hamster kidney cells and crystallized in both orthorhombic (P212121) and tetragonal (P41212) space groups. Both crystal forms diffract to high resolution (1.6 and 1.8 A, respectively) and have been solved by molecular replacement. Subsequent refinement resulted in final models for the structure of hTF/2N that had crystallographic R-factors of 18.1 and 19.7% for the two crystal forms, respectively; these models represent the highest-resolution transferrin structures determined to date. The hTF/2N polypeptide has a folding pattern similar to those of other transferrins, including the presence of a deep cleft that contains the metal-binding site. In contrast to other transferrins, both crystal forms of hTF/2N display disorder at the iron-binding site; model building suggests that this disorder consists of alternative conformations of the synergistically bound carbonate anion, the side chain for Arg-124, and several solvent molecules. Subsequent refinement revealed that conformation A has an occupancy of 0.63-0. 65 and corresponds to the structure of the iron-binding site found in other transferrins. The alternative conformation B has an occupancy of 0.35-0.37; in this structure, the carbonate has rotated 30 degrees relative to the iron and the side chain for Arg-124 has moved to accommodate the new carbonate position. Several water molecules appear to stabilize the carbonate anion in the two conformations. These structures are consistent with the protonation of the carbonate and resulting partial removal of the anion from the metal; these events would occur prior to cleft opening and metal release.

The relationship between structure and stability has been investigated for the folded forms and the unfolded forms of iso-2 cytochrome c and a variant protein with a stability-enhancing mutation, N52I iso-2. Differential scanning calorimetry has been used to measure the reversible unfolding transitions for the proteins in both heme oxidation states. Reduction potentials have been measured as a function of temperature for the folded forms of the proteins. The combination of measurements of thermal stability and reduction potential gives three sides of a thermodynamic cycle and allows prediction of the reduction potential of the thermally unfolded state. The free energies of electron binding for the thermally unfolded proteins differ from those expected for a fully unfolded protein, suggesting that residual structure modulates the reduction potential. At temperatures near 50 degrees C the N52I mutation has a small but significant effect on oxidation state-sensitive structure in the thermally unfolded protein. Inspection of the high-resolution X-ray crystallographic structures of iso-2 and N52I iso-2 shows that the effects of the N52I mutation and oxidation state on native protein stability are correlated with changes in the mobility of specific polypeptide chain segments and with altered hydrogen bonding involving a conserved water molecule. However, there is no clear explanation of oxidation state or mutation-induced differences in stability of the proteins in terms of observed changes in structure and mobility of the folded forms of the proteins alone.

Preliminary crystallographic study has been can•ied out with maltotetraose-forming amylase(E.C.3.2.1.60,G4-amylase), which was isolated and purified from Alcaligenes sp.found from Chinese soiL G4-amylase is a unique amylase which catalyzes the release of a-maltotetraose from the nonreducing ends of starch molecules.It is commercially important for producing maltotetraose with superior properties.This enzyme has molecule mass of about 60kD and pi4.45.After an intensive screening of crystallization conditions was conducted with the enzyme, better G4-amylase crystals could be obtained using the hanging drop method with a drop consisting of lOmg/ml enzyme sample solution and equal volume of reservoir solution containing O.lM Cacodylate buffer (pH6.5),0.2M calcium acetate and 18% PEG-8000.The G4-amylase crvstals are orthogonaL and the unit cell has dimensions a=46.6A,b~65.8A and c=l 70.9A and one molecule per asymmetJic unit 2.8A intensity data have been collected with a G4-amylase crystal on Mar Research IP detector system in our laboratory.Fmther structure detennination of G4-amylase is under way.

MFE-23 is a single-chain Fv (scFv) antibody ti•agment that was selected from a phage-display library for high affinity binding to carcinoembryonic antigen (CEA), and has important clinical uses in the detection, monitming and targetting of colon cancer.CEA is a seven-domain cell adhesion molecule which is expressed at high levels on colon carcinoma cells.150: 256 residues) consists of anN-terminal VH domain, a IS-residue linker, a VL domain and a 12-residue tag.MFE-23 was expressed in E. coli and purified on CEA coupled to Sepharose.Cr-ystals were produced by the hanging-drop method.MFE-23 at 2 mg/ml was mixed 1:1 with precipitant (100 mM Tris-HCI at pH 6.5 containing 45% saturated ammonium sulfate), and a 10 ~ll drop of this mixture was equilibrated against 0.5 ml of precipitant at l8°C.Well-fom1ed crystals fom1ed within several days, and diffracted to 0.2 nm resolution.Diffraction data were collected using an R-A.'CIS-ITC mounted on an RU200 rotating anode X -ray source.The crystal belongs to the trigonal space group P3221 and has unit cell dimensions of a= b = 6.170 nm and c = J 2.794 nm.A single crystal was used for structure detennination.Data to a resolution of 0.28 nm were processed using DENZO and the CCP4 program package.The structure was solved by molecular replacement using the Fv coordinates from a mmine IgA Fab fragment (Brookhaven code 2FBJ).The antibody complementarity determining regions, the linker and the tag were omitted for the calculation of initial 2Fo-Fc electron density maps.Refinement of the MFE-23 structure is cunently in progress, utilizing the rigid-body refinement and the positional refinement algorithms ofX-PLOR ar1d manual rebuilding of the model in 0. PS04.15.12 ARE "HOT SPOTS" [SUPER- FLEXIBLE REGIONS]

The coupled oxidation of protoheme IX at the active sites of heme proteins exposed to ascorbate in the presence of dioxygen has been known for over 65 years and is related to the catalytic conversion of protoheme SX to biliverdin by the enzyme heme oxygenase. The present report demonstrates that replacement of two residues present in the distal heme pocket of horse heart myoglobin (Mb), Val67 and Val68, with alanine and serine, respectively, increases the efficiency of coupled oxidation in the active site of myoglobin. HPLC analysis of the heme oxidation product demonstrates that the specificity of wild-type myoglobin for opening the heme ring at the alpha-meso carbon is retained by the variant. The infrared spectrum of the carbonyl derivative of the variant exhibits nu(CO) bands of comparable intensity at 1948 and 1958 cm(-1), which are consistent with the presence of the polar serine residue in close proximity to the bound ligand. The high-resolution crystal structure of the Va167Ala/Va168Ser metMb variant establishes that a hydrogen bond forms between the hydroxyl group of Ser68 and the coordinated water molecule. The increased rate of coupled oxidation exhibited by the variant is attributed to the increased polarity of the distal pocket created by the amino acid substitutions. These results are discussed in light of recent work concerning the active site of heme oxygenase.

The structure of human pancreatic alpha-amylase has been determined to 1.8 A resolution using X-ray diffraction techniques. This enzyme is found to be composed of three structural domains. The largest is Domain A (residues 1-99, 169-404), which forms a central eight-stranded parallel beta-barrel, to one end of which are located the active site residues Asp 197, Glu 233, and Asp 300. Also found in this vicinity is a bound chloride ion that forms ligand interactions to Arg 195, Asn 298, and Arg 337. Domain B is the smallest (residues 100-168) and serves to form a calcium binding site against the wall of the beta-barrel of Domain A. Protein groups making ligand interactions to this calcium include Asn 100, Arg 158, Asp 167, and His 201. Domain C (residues 405-496) is made up of anti-parallel beta-structure and is only loosely associated with Domains A and B. It is notable that the N-terminal glutamine residue of human pancreatic alpha-amylase undergoes a posttranslational modification to form a stable pyrrolidone derivative that may provide protection against other digestive enzymes. Structure-based comparisons of human pancreatic alpha-amylase with functionally related enzymes serve to emphasize three points. Firstly, despite this approach facilitating primary sequence alignments with respect to the numerous insertions and deletions present, overall there is only approximately 15% sequence homology between the mammalian and fungal alpha-amylases. Secondly, in contrast, these same studies indicate that significant structural homology is present and of the order of approximately 70%. Thirdly, the positioning of Domain C can vary considerably between alpha-amylases. In terms of the more closely related porcine enzyme, there are four regions of polypeptide chain (residues 237-250, 304-310, 346-354, and 458-461) with significantly different conformations from those in human pancreatic alpha-amylase. At least two of these could play a role in observed differential substrate and cleavage pattern specificities between these enzymes. Similarly, amino acid differences between human pancreatic and salivary alpha-amylases have been localized and a number of these occur in the vicinity of the active site.

A site-specific mutant of horse heart myoglobin has been prepared in which the distal heme pocket residue, His64, is replaced by tyrosine. The structure of this myoglobin variant has been determined to 2.0-A resolution using x-ray diffraction techniques and refined to a final crystallographic R-factor of 16.9%. The polypeptide backbone conformation of the His64-->Tyr variant of myoglobin is very similar to that of the wild-type protein. However, in the variant the water normally found coordinated to the heme iron atom and hydrogen-bonded to His64 has been displaced by the hydroxyl oxygen of the Tyr64 side chain. The tyrosine oxygen atom is directly coordinated to the heme iron atom with a bond length of 2.18 A. Distortion of heme planarity and changes in the packing of the Leu29 and Leu104 side chains are related to this mutation. The ligand environment of the ferric iron has been studied by electron paramagnetic resonance (EPR) spectroscopy using crystalline material and protein in solution. The protein in solution exhibits a rhombically split ferric high spin EPR spectrum with g values of 6.64, 5.34, and 1.98. The EPR spectrum of the crystalline sample consists of two different ferric high spin signals. The main signal is similar to the signal observed in solution and is assigned to His93-Fe(III)-Tyr64 coordination. The relatively high rhombicity of this signal can be explained as arising from distortions of the heme plane seen in the crystal structure. The second, more axial high spin signal found in the crystalline state can be tentatively assigned to another form of iron ligation with a different iron-tyrosine bond length and a less distorted heme plane.

Human pancreatic α-amylase has been isolated using a glycogen affinity precipitation procedure and crystallized in a form suitable for high resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P212121, with unit cell dimensions of a = 53·04 Å, b = 74·80 Å and c = 137·34 Å, and contain only one protein molecule per asymmetric unit. Diffraction data have been collected and found to extend to 1·6 Å resolution. These studies form the basis elucidating the full atomic structure of human pancreative α-amylase and thereby providing insight into the catalytic mechaism of this enzyme.

The N-terminal lobe of recombinant human serum transferrin (residues 1 to 337) has been crystallized in a form suitable for high-resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P212121, with unit cell dimensions of a = 44.9 Å, b = 57.0 Å and c = 135.9 Å, and diffract to beyond 2 Å resolution. Further studies show that isomorphous crystals of specifically designed mutants of this protein can also be grown. Structural studies of both recombinant and mutant protein forms will provide a basis for understanding the mechanism by which human serum transferrin functions.