The X-ray structure determination of bovine carbonmonoxy hemoglobin at 2.1 A resoultion and its relationship to the quaternary structures of other hemoglobin crystal froms

Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.     

The enigma of the liganded hemoglobin end state: a novel quaternary structure of human carbonmonoxy hemoglobin

The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the "classical" liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between beta2His97 and alpha1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T --> R3 and T --> R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the alpha-heme, reduced steric contact between the beta-heme ligand and the distal residues, smaller alpha- and beta-clefts, and reduced alpha1-alpha2 and beta1-beta2 iron-iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure-function relationships that are now based on T --> R, T -->R --> R2, or T --> R2 --> R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.     

Structure of a single amino acid mutant of aspartate carbamoyltransferase at 2.5-A resolution: implications for the cooperative mechanism

One of the many interactions important for stabilizing the T state of aspartate carbamoyltransferase occurs between residues Tyr240 and Asp271 within one catalytic chain. The functional importance of this polar interaction was documented by site-directed mutagenesis in which the tyrosine was replaced by a phenylalanine [Middleton, S. A., & Kantrowitz, E. R. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5866-5870]. In the Tyr240----Phe mutant, the aspartate concentration required to achieve half-maximum velocity is reduced to 4.7 from 11.9 mM for the native enzyme. Here, we report an X-ray crystallographic study of the Tyr240----Phe enzyme at 2.5-A resolution. While employing crystallization conditions identical with those used to grow cytidine triphosphate ligated T-state crystals of the native enzyme, we obtain crystals of the mutant enzyme that are isomorphous to those of the native enzyme. Refinement of the mutant structure to an R factor of 0.219 (only eight solvent molecules included) and subsequent comparison to the native T-state structure indicate that the quaternary, tertiary, and secondary structures of the mutant are similar to those for the native T-state enzyme. However, the conformation of Phe240 in one of the two crystallographically independent catalytic chains contained in the asymmetric unit is significantly different from the conformation of Tyr240 in the native T-state enzyme and similar to the conformation of Tyr240 as determined from the R-state structure [Ke, H.-M., Lipscomb, W. N., Cho, Y. J., & Honzatko, R. B. (1988) J. Mol. Biol. (in press)], thereby indicating that the mutant has made a conformational change toward the R state, localized at the site of the mutation in one of the catalytic chains.     

Mixed regioselectivity in the Arg-177 mutants of Corynebacterium diphtheriae heme oxygenase as a consequence of in-plane heme disorder

It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce approximately 30% delta-biliverdin [Zhou, H., et al. (2000) J. Am. Chem. Soc. 122, 8311-8312]. Two plausible mechanisms were proposed to explain the observations. (a) Electrostatic repulsion between E183 (D183) and one of the heme propionates forces the heme to rotate, thereby placing the delta-meso carbon in a position that is susceptible to oxidation. (b) Rearrangement of the distal pocket structure is triggered by the formation of a hydrogen bond between E183 (D183) and K179. A change in the pK(a) for the Fe(III)-H(2)O to Fe(III)-OH transition of the mutants was interpreted to be consistent with rearrangement of the hydrogen bond network in the distal pocket. The large similarities between the high-frequency portion of the (1)H NMR spectra corresponding to the wild type and R183E and R183D mutants were interpreted to indicate that the heme in the mutants is not rotated to a significant extent. We have re-examined this issue by studying the corresponding R177 mutants in heme oxygenase from Corynebacterium diphtheriae (cd-HO). Replacing R177 with E or D results in the formation of approximately 55% alpha- and 45% delta-biliverdin, whereas the R177A mutant retains alpha-regioselectivity. In addition, the K13N/Y130F/R177A triple mutant catalyzed the formation of 60% delta- and 40% alpha-biliverdin, while single mutants K13N and Y130F did not appreciably change the regioselectivity of the reaction. The pK(a) of the Fe(III)-H(2)O to Fe(III)-OH transition in wild-type cd-HO is 9.1, and those of the R177E, R177D, R177A, and K13N/Y130F/R177A mutants are 9.4, 9.5, 9.2, and 8.0, respectively. Thus, no obvious correlation exists between the changes in pK(a) and the altered regioselectivity. NMR spectroscopic studies conducted with the R177D and R177E mutants of cd-HO revealed the presence of three heme isomers: a major (M) and a minor (m) heme orientational isomer related by a 180 degrees rotation about the alpha-gamma meso axis and an alternative seating (m') which is related to m by an 85 degrees in-plane rotation of the macrocycle. The in-plane rotation of m to acquire conformation m' is triggered by electrostatic repulsion between the side chains of D or E at position 177 and heme propionate-6. As a consequence, the delta-meso carbon in m' is placed in the position occupied by the alpha-meso carbon in m, where it is susceptible to hydroxylation and subsequent formation of delta-biliverdin.     

Intersubunit hydrogen bond acts as a global molecular switch in Escherichia coli aspartate transcarbamoylase

Tyr 165 in the catalytic subunit of Escherichia coli aspartate transcarbamoylase (ATCase, EC 2.1.3.2) forms an intersubunit hydrogen bond in the T state with Glu 239 in the 240s loop of a second catalytic subunit, which is broken in the T to R transition. Substitution of Tyr 165 by Phe lowers substrate affinity by approximately an order of magnitude and alters the pH profile for enzyme function. We have determined the crystal structure of Y165F at 2.4 Å resolution by molecular replacement, using a wild-type T state structure as the probe, and refined it to an R value of 25.2%. The Y165F mutation induces a global conformational change that is in the opposite direction to the T to R transition and therefore results in an extreme T state. The two catalytic trimers move closer by ∼0.14 Å and rotate by ∼0.2°, in the opposite direction to the T→R rotation; the two domains of each catalytic chain rotate by ∼2.1°, also in the opposite direction to the T→R transition; and the 240s loop adopts a new conformation. Residues 229 to 236 shift by ∼2.4 Å so that the active site is more open. Residues 237 to 244 rotate by ∼24.1°, altering interactions within the 240s loop and at the C1-C4 and C1-R4 interfaces. Arg 167, a key residue in domain closure and interactions with L-Asp, swings out from the active site to interact with Tyr 197. This crystal structure is consistent with the functional properties of Y165F, expands our knowledge of the conformational repertoire of ATCase, and indicates that the canonical T state does not represent an extreme. Proteins 33:430–443, 1998. © 1998 Wiley-Liss, Inc.     

Quaternary structure of carbonmonoxyhemoglobins in solution: structural changes induced by the allosteric effector inositol hexaphosphate

We have applied the residual dipolar coupling (RDC) method to investigate the solution quaternary structures of (2)H- and (15)N-labeled human normal adult recombinant hemoglobin (rHb A) and a low-oxygen-affinity mutant recombinant hemoglobin, rHb(alpha96Val-->Trp), both in the carbonmonoxy form, in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using a stretched polyacrylamide gel as the alignment medium. Our recent RDC results [Lukin, J. A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517-520] indicate that the quaternary structure of HbCO A in solution is a dynamic ensemble between two previously determined crystal structures, R (crystals grown under high-salt conditions) and R2 (crystals grown under low-salt conditions). On the basis of a comparison of the geometric coordinates of the T, R, and R2 structures, it has been suggested that the oxygenation of Hb A follows the transition pathway from T to R and then to R2, with R being the intermediate structure [Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113-11117]. The results presented here suggest that IHP can shift the solution quaternary structure of HbCO A slightly toward the R structure. The solution quaternary structure of rHbCO(alpha96Val-->Trp) in the absence of IHP is similar to that of HbCO A in the presence of IHP, consistent with rHbCO(alpha96Val-->Trp) having an affinity for oxygen lower than that of Hb A. Moreover, IHP has a much stronger effect in shifting the solution quaternary structure of rHbCO(alpha96Val-->Trp) toward the R structure and toward the T structure, consistent with IHP causing a more pronounced decrease in its oxygen affinity. The results presented in this work, as well as other results recently reported in the literature, clearly indicate that there are multiple quaternary structures for the ligated form of hemoglobin. These results also provide new insights regarding the roles of allosteric effectors in regulating the structure and function of hemoglobin. The classical two-state/two-structure allosteric mechanism for the cooperative oxygenation of hemoglobin cannot account for the structural and functional properties of this protein and needs to be revised.     

Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state

Solution scattering curves evaluated from the crystal structures of the T and R states of the allosteric enzyme aspartate transcarbamylase from Escherichia coli were compared with the experimental x-ray scattering patterns. Whereas the scattering from the crystal structure of the T state agrees with the experiment, large deviations reflecting a significant difference between the quaternary structures in the crystal and in solution are observed for the R state. The experimental curve of the R state was fitted by rigid body movements of the subunits in the crystal R structure which displace the latter further away from the T structure along the reaction coordinates of the T→R transition observed in the crystals. Taking the crystal R structure as a reference, it was found that in solution the distance between the catalytic trimers along the threefold axis is 0.34 nm larger and the trimers are rotated by 11° in opposite directions around the same axis; each of the three regulatory dimers is rotated by 9° around the corresponding twofold axis and displaced by 0.14 nm away from the molecular center along this axis. Proteins 27:110–117 © 1997 Wiley-Liss, Inc.     

A survey of hemoglobin quaternary structures

We perform an analysis of the quaternary structure and dimer/dimer interface in the crystal structures of 165 human hemoglobin tetramers; 112 are in the T, 17 the R, 14 the Y (or R2) state; 11 are high-affinity T state mutants, and 11 may either be intermediates between the states, or off the allosteric transition pathway. The tertiary structure is fixed within each state, in spite of the different ligands, mutations, and chemical modifications present in individual entries. The geometry of the tetramer assembly is essentially the same in all the R or the Y state entries; it is slightly different in high salt and low salt crystals of T state hemoglobins. The dimer/dimer interface differs in terms of size, chemical composition and polar interactions, between the states. It is loosely packed, like crystal packing contacts or the subunit interface of weakly associated homodimers, and unlike most oligomeric proteins, which have close-packed interfaces. The loose packing is most obvious in the liganded forms, where the tetramer is known to dissociate at low concentration. We identify cavities that contribute to the loose packing of the α1β2 and α2β1 contacts. Two pairs of cavities occur recurrently in both the T and the R state tetramers. They may contribute to the allosteric mechanism by facilitating the subunit movements and the tertiary structure changes that accompany the transition from T to R to Y.     

Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP

The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP have been solved at 2.9 A, 2.9 A and 2.2 A resolution, respectively. The structure of R state GPa is nearly identical to the structure of sulphate-activated R state GPb, except in the region of Ser14, where there is a covalently attached phosphate group in GPa and a non-covalently attached sulphate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer are similar to those observed previously for T state GPa. The quaternary and tertiary structural changes on the T to R transition allow these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure results in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg10, the first residue to be located from the N terminus, in R state GPa and T state GPb is 50 A. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix alpha 2 and the cap' (residues 35' to 45' that form a loop between the 1st and 2nd alpha helices, alpha 1' and alpha 2' of the other subunit. The prime denotes residues from the other subunit). The interactions made by the N-terminal residues induce structural changes at the cap'/alpha 2 helix interface that lead to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifts 1.2 to 2.1 A for residues 35 to 45) are partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes are between 0.5 and 1.3 A. AMP binds to R state GPb with at least 100-fold greater affinity and exhibits four additional hydrogen bonds, stronger ionic interactions and more extensive van der Waals' interactions with 116 A2 greater solvent accessible surface area buried compared with AMP bound to T state GPb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of T state aspartate carbamoyltransferase of the hyperthermophilic archaeon Sulfolobus acidocaldarius

Aspartate carbamoyltransferase (ATCase) is a model enzyme for understanding allosteric effects. The dodecameric complex exists in two main states (T and R) that differ substantially in their quaternary structure and their affinity for various ligands. Many hypotheses have resulted from the structure of the Escherichia coli ATCase, but so far other crystal structures to test these have been lacking. Here, we present the tertiary and quaternary structure of the T state ATCase of the hyperthermophilic archaeon Sulfolobus acidocaldarius (SaATC(T)), determined by X-ray crystallography to 2.6A resolution. The quaternary structure differs from the E.coli ATCase, by having altered interfaces between the catalytic (C) and regulatory (R) subunits, and the presence of a novel C1-R2 type interface. Conformational differences in the 240 s loop region of the C chain and the C-terminal region of the R chain affect intersubunit and interdomain interfaces implicated previously in the allosteric behavior of E.coli ATCase. The allosteric-zinc binding domain interface is strengthened at the expense of a weakened R1-C4 type interface. The increased hydrophobicity of the C1-R1 type interface may stabilize the quaternary structure. Catalytic trimers of the S.acidocaldarius ATCase are unstable due to a drastic weakening of the C1-C2 interface. The hyperthermophilic ATCase presents an interesting example of how an allosteric enzyme can adapt to higher temperatures. The structural rearrangement of this thermophilic ATCase may well promote its thermal stability at the expense of changes in the allosteric behavior.     

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.     

Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.     

Cooperativity and high pressure: stabilization of the R conformation of the allosteric aspartate transcarbamylase under the influence of pressure.

The allosteric enzyme aspartate transcarbamylase (ATCase) from E. coli shows homotropic cooperative interactions between its six catalytic sites for the binding of the substrate aspartate. This cooperativity is explained by the transition of the enzyme from a conformation which has a low affinity for aspartate (T state) to a conformation with high affinity (R state). The crystallographic structures of these two conformations are known to a resolution of 2.5 A and 2.1 A, respectively, and they reveal an important difference in the quaternary structure of the protein. Enzyme kinetics under high pressure were used to study the transition between the two states. It appears that in the presence of a low concentration of aspartate, conditions under which the enzyme is essentially in the T state, pressure promotes the transition to the R state, the maximal effect being observed at 120 MPa. This transition is accompagnied by a significant deltaV. This observation is in accordance with the change in the protein surface exposed to the solvent, and with the increased number of water molecules bound to the protein. Since the partial specific volume of the enzyme does not change significantly during the T to R transition, the negative deltaV is only related to the change in hydration of the protein. This result emphasizes a significant role of the protein-solvent interactions in this important regulatory conformational change.     

Coarse‐grained and all‐atom modeling of structural states and transitions in hemoglobin

Hemoglobin (Hb), an oxygen‐binding protein composed of four subunits (α1, α2, β1, and β2), is a well‐known example of allosteric proteins that are capable of cooperative ligand binding. Despite decades of studies, the structural basis of its cooperativity remains controversial. In this study, we have integrated coarse‐grained (CG) modeling, all‐atom simulation, and structural data from X‐ray crystallography and wide‐angle X‐ray scattering (WAXS), aiming to probe dynamic properties of the two structural states of Hb (T and R state) and the transitions between them. First, by analyzing the WAXS data of unliganded and liganded Hb, we have found that the structural ensemble of T or R state is dominated by one crystal structure of Hb with small contributions from other crystal structures of Hb. Second, we have used normal mode analysis to identify two distinct quaternary rotations between the α1β1 and α2β2 dimer, which drive the transitions between T and R state. We have also identified the hot‐spot residues whose mutations are predicted to greatly change these quaternary motions. Third, we have generated a CG transition pathway between T and R state, which predicts a clear order of quaternary and tertiary changes involving α and β subunits in Hb. Fourth, we have used the accelerated molecular dynamics to perform an all‐atom simulation starting from the T state of Hb, and we have observed a transition toward the R state of Hb. Further analysis of crystal structural data and the all‐atom simulation trajectory has corroborated the order of quaternary and tertiary changes predicted by CG modeling. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Quaternary structure and spin-state transition in azide methemoglobin A

The temperature-dependent ultraviolet and visible absorption changes of human azide methemoglobin with and without inositol hexaphosphate (IHP) were examined in a 4'-35 degrees C range. The 537-nm absorption change of IHP-free hemoglobin was about 1.2-fold larger than that of IHP-bound hemoglobin. The data were analyzed by considering the thermal spin equilibrium within the R and T conformers and the quaternary equilibrium between the two conformers. The spin equilibrium analysis suggested that the T conformer has a larger high-spin content than the R conformer. The quaternary equilibrium analysis, on the other hand, showed that the T conformer is more populated at lower temperature. The thermodynamic values for the quaternary equilibrium were determined to be delta H = -13.3 kcal/mol and delta S = -47.6 eu. The large negative delta H and delta S values were compensated for each other to give a small energy difference between the two quaternary states, e.g., delta G4 = 670 cal/mol of tetramer at 20 degrees C. The coincidence of the temperature-dependent IHP-induced changes in the visible and ultraviolet absorptions of heme and aromatic chromophores at the subunit boundaries suggested that the quaternary transition energy is not localized at heme moiety. The reverse temperature dependence of the T conformer fraction as compared with the high-spin fraction of heme iron was interpreted as indicating that the appearance of the T state is not directly coupled with an increase in the strain of Fe-N(F8 His) linkage in azide methemoglobin A.     

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.     

Picosecond dynamics of T and R forms of aspartate transcarbamylase: a neutron scattering study

E. coli aspartate transcarbamylase (ATCase) is a 310 kDa allosteric enzyme which catalyses the first committed step in pyrimidine biosynthesis. The binding of its substrates, carbamylphosphate and aspartate, induces significant conformational changes. This enzyme shows homotropic cooperative interactions between the catalytic sites for the binding of aspartate. This property is explained by a quaternary structure transition from T state (aspartate low affinity) to R state (aspartate high affinity) accompanied by a 5% increase of radius of gyration of ATCase. The same quaternary structure change is observed upon binding of the bisubstrate analogue PALA (N-(phosphonacetyl)-L-aspartate. Owing to the large incoherent neutron scattering cross-section of the hydrogen atom and the abundance of this element in proteins, inelastic neutron scattering gives a global view of protein dynamics as sensed via the individual motions of its hydrogen atoms. We present neutron scattering results of the local dynamics (few angstroms), at short time (few tens of picoseconds), of ATCase in T and R forms. Compared to the T form, we observe an increased mobility of the protein in the R form that we associate to an increase of accessible surface area to the solvent. Beyond this specific result, this highlights the key role of the accessible surface area (ASA) in dynamic contribution to inelastic neutron data in the picosecond time scale. In particular, we want to stress out (i) that a difference at the picosecond time scale does not allow to conclude to a difference in the dynamics at a longer time scale and to address whether the T state is looser than the R state (ii) how challenging is, any comparison in terms of general dynamics (tense or relaxed) between dynamic values deduced from experimental neutron data on proteins with different sequences and therefore ASA. This caveat holds particularly when comparing dynamics of a mesophile with the corresponding extremophile.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Allosteric interpretation of the oxygen-binding reaction of human hemoglobin tetramers

An allosteric model is presented that provides a simple explanation for the low population of triply ligated species, relative to the other species, in the oxygenation of human hemoglobin tetramers as found in high-concentration studies [Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry (preceding paper in this issue)]. The model is a quantitative interpretation of the Perutz mechanism [Perutz, M. F. (1970) Nature (London) 228, 726-739] and is based on a number of structural and thermodynamic findings so far reported in the analysis of hemoglobin properties. Human hemoglobin is assumed to exist in two quaternary states: the T or low-affinity state and the R or high-affinity state. An extreme chain heterogeneity in the T state is postulated so that oxygen binds only to the alpha chains. Nearest-neighbor interactions between the alpha chains may lead to cooperativity within the T state. The R state is noncooperative, and both the alpha and beta chains have equal oxygen affinity.