Demetrius Tsernoglou

Temperature-dependent X-ray diffraction as a probe of protein structural dynamics

X-ray diffraction at four temperatures from 220 to 300 K coupled with crystallographic refinement yields the mean-square displacements and conformational potentials of all 1,261 non-hydrogen atoms of metmyoglobin. The results are interpreted to indicate a condensed core around the haem, semi-liquid regions towards the outside and a possible pathway for ligands. It is concluded that X-ray diffraction can provide the spatial distribution of the dynamic features of a protein.

The crystal structure of a post-synaptic neurotoxin from sea snake at 2.2 Å resolution

Among the most lethal proteins in the venoms of sea snakes and elapid land snakes are the post-synaptic neurotoxins. These act by blocking the nicotinic acetylcholine receptor of the muscle motor end plate [ 1 ] , thus preventing transmission across the cholinergic synapse. Because of their extremely specific and tight binding to the receptor (KD is on the order of lo-“), these toxins have been the object of intensive chemical study [2], and have also been used to isolate the receptor protein [3,4] . Up to now there has been no information on the three-dimensional structure of the neurotoxins to aid in understanding their mode of action. We have solved the crystal structure of a neurotoxin from the sea snake Laticauda semifasciata from the Philippines Sea. This is a ‘short’ neurotoxin of 62 amino acids cross-linked by four disulfide bridges [S] . The structure has been determined at 2.2 A resolution. The molecule is a disc with one extended loop containing most of the residues believed essential for toxicity. We conclude that this structure is common to all snake venom neurotoxins, and that the postsynaptic toxins act by inserting the loop into a cleft or channel in the acetylcholine receptor.

Structure of oxidized flavodoxin from Anacystis nidulans

The structure of oxidized flavodoxin from the cyanobacterium Anacystis nidulans has been determined at 2.5 A resolution with phases calculated from ethylmercury phosphate and dimercuriacetate derivatives. The determination of partial sequences, including a total of 85 residues, has assisted in the interpretation of the electron density. Preliminary refinement of a partial model (1072 atoms) has reduced R to 0.349 for the 10.997 reflections between 2.0 and 5.0 A with 1 greater than 2 sigma. The polypeptide backbone, which comprises 167 residues in the current model, adopts the familiar beta-alpha-beta conformation found in other flavodoxins and in the nucleotide-binding domains of the pyridine-nucleotide dehydrogenases, with five parallel strands in the central sheet. Comparison with flavodoxin from Clostridium MP (138 residues) shows that extra residues of A. nidulans flavodoxin are accommodated in a major insertion about 20 residues in length, which forms a lobe adjacent to the fifth strand of parallel sheet, and in additions to several external segments. Residues added between the fourth sheet strand and the start of the third helix alter the environment of the pyrimidine end of the flavin mononucleotide ring. The flavin mononucleotide phosphate binds to the start of helix 1, interacting with hydroxyamino acids and with main-chain amide groups. Two hydrophobic residues, both tentatively identified as Trp, enclose the isoalloxazine ring; the solvent-exposed Trp is nearly parallel to the flavin ring. The hydrophobic environment provided by these residues must be partly responsible for the pronounced vibrational resolution of the flavin spectrum near 450 nm. The flavin ring is tilted relative to its orientation in Clostridium MP flavodoxin. In addition, atoms N-3 and O-2 alpha of the isoalloxazine appear to form hydrogen bonds to the backbone at CO97 and NH99 in a conformation entirely different from that found in Clostridium MP flavodoxin but structurally analogous to Desulfovibrio vulgaris flavodoxin.

The three-dimensional structure of the complex of proteinase K with its naturally occurring protein inhibitor, PKI3

Proteinase K forms a 1:1 stable complex with its naturally occurring protein inhibitor, PKI3. The crystal structure of this complex has been determined by a combination of molecular replacement and single isomorphous replacement methods. The model comprises all of the 459 residues: 279 for proteinase K and 180 for PKI3, and it was refined to an R‐factor of 19.2% at a resolution of 2.5 Å. Association of these two molecules in the complex indicates the binding of PKI3 in the substrate recognition site of the enzyme. The active serine residue of proteinase K in this complex possesses a somewhat different configuration to that found in its native structure and hence renders the enzyme inactive.

New high pH crystal forms of ribonucleases A and S.

Bovine pancreatic ribonuclease A has been crystallized at pH 9 from (NH 4 ) 2 SO 4 /CsCl. The crystals (designated form M) belong to the trigonal space group P 3 1 21 and have unit cell dimensions a =64·34 A, c =65·00 A, ψ=120°. They are isomorphous with crystal form W of bovine pancreatic ribonuclease S, which is grown at pH 6·6. Ribonuclease S has also been crystallized at pH 9 and is isomorphous with forms M and W. Significant intensity differences exist among the crystal forms, suggesting pH-induced conformational changes at least as large as those caused by limited proteolysis. The crystals of form M diffract well to at least 2·8 A resolution.

The structure of subtilopeptidase A: I. X-ray crystallographic data

Crystals of subtilopeptidase A have been studied by means of X-ray diffraction. They are orthorhombic, space group P212121 with a = 76 73 A, b = 55.35 Aand c = 53.00 A. There is one molecule per asymmetric unit and the solvent content is 42%. The crystals diffract very well and appear suitable for detailed study of the three-dimensional structure of the enzyme.

ELECTRON TRANSPORT SYSTEM FOR ADRENOCORTICAL MITOCHONDRIAL STEROID HYDROXYLATION REACTIONS: THE MECHANISM OF THE HYDROXYLATION REACTIONS AND PROPERTIES OF THE FLAVOPROTEIN-IRON-SULFUR PROTEIN COMPLEX*

It has been established by o~rselves-~ and others- that the electron transport system for steroid 1 IP-, 18-, and cholesterol side-chain-cleaving hydroxylation reactions of adrenocortical mitochondria consists of a flavoprotein (adrenodoxin reductase), an iron-sulfur protein (adrenodoxin), and a cytochrome (P-450). This electron transport system is believed to be located in the inner mitochondria1 membrane and is not directly coupled with the transport system for oxidative phosphorylation, except at the level of NADH-NADP transhydrogenase. A similar or identical electron transport system is known to exist in testis and ovary. In brief summary, adrenodoxin has a molecular weight of 12,500, consisting of 114 amino acid residues, and it contains 2 g atoms of iron and 2 mol labile sulfur per mol protein. Adrenodoxin reductase has a molecular weight of 54,000, consisting of one FAD per mol protein. The molecular characteristics of P-450 have not yet been established, due to difficulties is its solubilization and purification. In spite of considerable amounts of information concerning these redox components, the mechanism of the hydroxylation of steroids by the P-450 system remains largely unknown. In this report, we wish to present our recent findings on the molecular oxygen activation mechanisms in steroid hydroxylation reactions, and on the nature of the interaction between flavoprotein and ironsulfur protein.

Protein sequencing by computer graphics

A computer graphics system has been used to fit a putative sequence to an electron density map of a sea snake neurotoxic protein at 2.2 A resolution. The complete sequence of this small protein could be determined from the map with very little ambiguity. In two places probable errors in the published chemical sequence were detected. This is the first instance in which a complete three-dimensional structure was solved with the use of computer graphics alone, without the construction of a physical model.

Preliminary X-ray crystallographic data for the semisynthetic non-covalent complex of residues 1 to 118 and 111 to 124 of bovine pancreatic ribonuclease

Abstract The enzymically active, semisynthetic complex formed by residues 1 through 118 and residues 111 through 124 of bovine pancreatic ribonuclease has been crystallized at pH 5.7 from ( NH 4 ) 2 SO 4 CsCl solutions. The crystals belong to space group P3221, have unit cell dimensions a and b = 64.4 A , c = 64.5 A , and γ = 120° and are isomorphous with form M of ribonuclease A as well as forms W and R of ribonuclease S. They diffract well and may be expected to yield a structure defined to at least 3.0 A resolution.