Identification and analysis of extended strands and β-sheets in globular proteins

A method to identify β-sheets in globular proteins from extended strands, using only α-carbon positions, has been developed. The strands that form β-sheets are picked up by means of simple distance criteria. The method has been tested by applying it to three proteins with accurately known secondary structures. It has also been applied to ten other proteins wherein only α-carbon coordinates are available, and the list of β-sheets obtained. The following points are worth noting: (i) The sheets identified by the algorithm are found to agree satisfactorily with the reported ones based on backbone hydrogen bonding, wherever this information is available. (ii) β-Strands that do not form parts of any sheet are a common feature of protein structures. (iii) Such isolated β-strands tend to be short. (iv) The conformation corresponding to the preferred right-handed twist of the sheet is overwhelmingly observed in both the sheet-forming and isolated β-strands.     

A new motif for inhibitors of geranylgeranyl diphosphate synthase

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure–function relationship which is dependent on the nature of the alkyl group at the α-carbon.     

Visualization of structural similarity in proteins

Two new methods for the visualization of structural similarity in proteins with known three-dimensional structures are presented. They are based on the degree of equivalence of α-carbon pairs in two proteins. The quantitative measure for residue equivalence is the comparison score generated using the sequence and structure alignment method of Taylor and Orengo, which is based on the comparison of interatomic distances (and other properties that can be defined on a residue basis).The first method uses information on corresponding α-carbon positions to display vectors joining these structurally equivalent residues. These vectors can be defined as target constraints, and their minimization “bends” the two proteins toward a common average structure. In the average structure the corresponding residues virtually superpose, while insertions and deletions become clearly visible.The second method uses the comparison scores to perform a weighted least-squares fit of the two structures. It is further used to color code the two structures according to the score value, i.e., their similarity, on a continuous scale from red to blue. Examples of the methods for the comparison of flavodoxin, chemotaxis Y protein and L-arabinose-binding protein are given.     

Application of distance geometry to the proton assignment problem

Assignment of the hydrogen spectrum is the first step in the conventional procedure for the determination of molecular structure by ¹H-nmr. In this paper, we explore the possibility of directly exploiting the distances derived from nuclear Overhauser effect experiments to generate a three-dimensional structure that is then assigned based on knowledge of the connectivity or primary sequence. This effort is analogous to that of the protein crystallographers in tracing electron density of the peptide chain. In particular, we compare structures produced by distance geometry to known peptide secondary structures to see what level of information is required to “trace” the backbone α-carbon and amide hydrogens and the β-carbon hydrogens. We conclude that this approach is only useful with excellent quality stereo-resolved data. © 1993 John Wiley & Sons, Inc.     

On the mechanism of formation of arterenone in insect cuticular hydrolyzates

Arterenone (2-amino-3′,4′-dihydroxy acetophenone) is an important hydrolytic product generated from lightly colored sclerotized cuticle that use N-acyldopamine derivatives for crosslinking reactions. It seems to arise from 1,2-dehydro-N-acetyldopamine (dehydro NADA) that has been crosslinked to the cuticular components. However, the mechanism of generation of arterenone, which has two protons on the α-carbon and no proton on the β-carbon atom from dehydro NADA crosslinks that have one proton each on these two side chain carbons, remained elusive and undetermined. To investigate the mechanism of this transformation, we synthesized specifically labeled β-deuterated dehydro NADA and incubated with Sarcophaga bullata cuticle undergoing larval puparial transformation. We also isolated the dimeric products formed during the tyrosinase-mediated oxidation of dehydro NADA. Hydrolysis of both β-deuterated dehydro NADA treated cuticle and β-deuterated dehydro NADA dimer generated arterenone as the major hydrolytic product. Liquid chromatography-mass spectrometric analysis of this arterenone revealed the retention of deuterium from the β-position of dehydro NADA at the α-carbon atom of arterenone. Hydrolysis of β-deuterated dehydro NADA also generated the labeled arterenone under oxidative conditions, but not under anaerobic conditions. These results indicate the unique hydride shift from β-carbon to α-carbon during acid hydrolysis and reveal the mechanism of liberation of arterenone and related compounds from dehydro NADA linked cuticle.     

Chirality and Handedness of Protein Structures

In proteins, the polypeptide chain forms a number of right-and left-handed helices and superhelices, right-and left-turned hairpins, and some other structures that are nonsuperimposable, although they are not mirror images of each other as the Lamino acids are not converted to the Damino acids. This property of protein structures will be referred to here as pseudo-chirality–or handedness. It has been shown that there are two kinds of handedness in proteins–helical handedness and handedness of arrangement. Some protein structures exhibit both the kinds of handedness. Handedness is observed at all levels of protein structural organization–from α-helices, β-strands, hairpins, βαβ-units up to complex structural motifs, superhelices, and supramolecular structures in fibrous and polymer proteins. There are several structures that have unique handedness in proteins, for example, α-helices, αα-corners, βαβ-units, abcd-units, and so on. This property of the polypeptide chain is of particular value in protein folding and protein modeling, because it drastically reduces the number of possible folds.     

A nuclear magnetic resonance study of the helix–coil transition of poly(L-glutamic acid)

There have been many reports that the nuclear magnetic resonance (nmr) spectra of a large number of polypeptides exhibit peak doubling of the α-carbon and the α-carbon proton in the helix–coil transition region. One apparent exception to this generalization has been polypeptides with ionizable side chains, where the helix–coil transition is induced by changes in pH in aqueous solution. Because it is important to establish the proper theoretical reason for the peak doubling and its relation to the rate of conformational change of amino acid residues, we have reexamined the proton and carbon-13 nmr spectra, at high field, for two polydisperse samples of poly(L -glutamic acid). Doubling of the α-carbon proton resonance as well as those of the α- and β-carbon, and backbone carbonyl are observed for a low-molecular-weight sample (DP = 54), while a higher molecular weight sample (DP = 309), exhibits only single resonances. Thus, polydispersity by itself is not sufficient to observe peak doubling; low-molecular weight is also required.     

Catalytic asymmetric synthesis of cyclic α-alkyl-amino acid derivatives having a tetrasubstituted α-carbon

Catalytic asymmetric synthesis of various cyclic α-alkyl-amino acid derivatives having a tetrasubstituted α-carbon has been accomplished by the utilization of phase-transfer alkylation of α-alkyl-amino acid derivatives.     

Simple protein model building tool

An interactive protein model building program, named Alpha, running on the Evans & Sutherland PS340, is presented. It has two prominent features: flexible construction and an informative display of the protein model. These characteristics arise from the adoption and analysis of the α-carbon representation of a protein. Although its concept and program are simple. Alpha is a useful tool for investigation of the 3D structure of a protein, whether or not it is elucidated.     

The effects of pH on the rates of isotope exchange catalyzed by alanine aminotransferase.

Alanine aminotransferase catalyzes exchange of the β-hydrogens of alanine with the solvent at a rate commensurate with the rate of exchange of the α-hydrogen. These methyl protons are lost sequentially and intermediates having protons on the α-carbon but deuterium on the β-carbon were detected by nuclear magnetic resonance. The overall rates of exchange of both α-hydrogen and β-hydrogen were less than the rate of transamination and did not vary from pH 6–8. The α-hydrogen of glutamate, on the other hand, was found to exchange at a greater rate than the overall transamination rate with ketoglutarate. However the β-hydrogens of glutamate are not removed during the enzymic reaction. It is concluded that a basic group on the enzyme removes the proton from the α-carbon of alanine at a rate at least as great as the rate of transamination. Because the proton is held on the enzyme, it appears to exchange more slowly in alanine. Labilization of the α-hydrogen of amino acids does not appear to be the ratelimiting reaction of alanine aminotransferase, but occurs at a rate comparable to that of the overall reaction.     

Detecting circular and rectangular particles based on geometric feature detection in electron micrographs

Accurate and automatic particle detection from cryo-electron microscopy (cryo-EM images) is very important for high-resolution reconstruction of large macromolecular structures. In this paper, we present a method for particle picking based on shape feature detection. Two fundamental concepts of computational geometry, namely, the distance transform and the Voronoi diagram, are used for detection of critical features as well as for accurate location of particles from the images or micrographs. Unlike the conventional template-matching methods, our approach detects the particles based on their boundary features instead of intensities. The geometric features derived from the boundaries provide an efficient way for locating particles quickly and accurately, which avoids a brute-force searching for the best position/orientation. Our approach is fully automatic and has been successfully applied to detect particles with approximately circular or rectangular shapes (e.g., KLH particles). Particle detection can be enhanced by multiple sets of parameters used in edge detection and/or by anisotropic filtering. We also discuss the extension of this approach to other types of particles with certain geometric features.     

Intramolecular conformational transitions "random coil-helix-folded structure" in polypeptides.

A general theory has been developed for conformational intramolecular transitions in a single macromolecule with a high degree of polymerization (an infinite length model) capable of forming two types of ordered structures: the α-helix and the folded β-structure, as well as acquiring the random coil conformation. The phase diagram analysis of this system has shown that the regular β-structure state is separated from all other states of the chain by the phase boundary line. Any intersection of the phase boundary is a phase transition which can be either of the first order or second order, depending on values of the energy parameters of the system. Mechanisms of intramolecular rearrangements: β-structure–random coil and α-helix–β-structure have been discussed. It has been shown that there exist two different mechanisms for each of these rearrangements, and the regions of parameter variation corresponding to each mechanism have been specified.     

High-Level Expression of Rat Farnesyl:Protein Transferase inEscherichia colias a Translationally Coupled Heterodimer

Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the α- and β-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/LEscherichia coli.Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The β-subunit-coding sequence was placed upstream of the α-subunit coding sequence linked by overlapping β-subunit stop and α-subunit start codons. Additionally, the initial 88 codons of the α-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins inE. coli.Since previous attempts at recombinantly expressing FPTase inE. colifrom a translationally coupled system have demonstrated that initiation of translation of the α-subunit is poor, we propose that the optimization of the codons at the start of the α-subunit gene leads to the observed high level of recombinant expression.     

Alcohol Induced Reversal of Enantioselectivity in a Lipase Catalyzed Resolution of 2-Chloropropionic Acid

Alcohol induced reversal of enantioselectivity in the esterification of 2-chloropropionic acid using lipase from Candida cylindracea has been investigated. It was found that an alcohol having substituents both at the α- and the β-carbon preferentially esterified the S-acid, while a straight chain alcohol preferentially esterified the R-acid. Intermediate enantioselectivities were obtained with alcohols having substituents either at the α- or the β-carbon, but still in favor of the R-acid. An acyl binding domain composed of three subsites is proposed for this lipase; one for the hydrocarbon chain, a second for a methyl substituent and a third for an electronegative substituent.     

Stereochemical studies on cyclic peptides. IX. Conformational studies on cyclic tetrapeptides containing alternating cis and trans peptide units

Conformational analyses of cyclic tetrapeptides consisting of alternating cis and trans peptide units have been made using contact criteria and energy calculations. This study has been restricted to those structures having a symmetry element in the backbone ring, such as a twofold axis (d) or a center of inversion (i). There are five main results. (1) There are two distinct types of conformations, which are stereochemically favorable corresponding to each of twofold and inversion-symmetrical structures, designated as d₁, d₂ (for twofold symmetrical) and i₁, i₂ (for inversion-symmetrical). Among these, the i₁ type has the lowest energy when glycyl residues occur at all four α-carbon atoms. (2) With the glycyl residue at all four α-carbon atoms, methyl substitution at the cis peptide nitrogen atoms is possible in all the four types, whereas the substitution at trans peptide nitrogen atoms is possible only for the i₁ type. Thus only in the i₁ type can all the nitrogen atoms be methylated simultaneously. The conformation of the molecule in the crystal structure of cyclotetrasarcosyl belongs to the i₁ type. (3) When alanyl residues occur at all four α-carbon atoms, the possible symmetrical type is dependent on the enantiomorphic form and the actual sequence of the alanyl residues. (4) The methyl substitution at peptide nitrogen atoms for cyclic tetrapeptides having alanyl residues causes more stereochemical restriction in the allowed conformations than with glycyl residues. (5) The prolyl residue can be incorporated favorably at the cis-trans junction of both d and i types of structures. The results of the present study are compared with the data on cyclic tetrapeptides available from the crystal structure and nmr studies. The results show an overall agreement both regarding the type of symmetry and the conformational parameters.     

Automated interpretation of electron density maps as applied to Bence-Jones protein Rhe

Previous results obtained from the computerized interpretation (Greer, 1976) of a 3.0 Å electron density map of Bence—Jones protein Rhe have been compared with results (Wang et al., 1979) obtained by classical Richards' box techniques (Richards, 1968). Although the overall agreement between the two models is generally good, the computerized results contain several significant errors in the assignment of α-carbon atoms, which in our opinion are unlikely to be corrected without using human intervention together with other methods. However this test does show that the computerized method has potential.     

Nonradioactive immunoquantification of α-l-fucosidase protein in human colon tissues

α-l-Fucosidase is a glycosidase involved in the degradation of fucoglycoconjugates and has a diagnostic significance because it has been described to be altered in several known diseases. However, in vitro studies on enzymatic activities may not reflect the real protein levels in tissues. This paper describes a simple method to quantify α-l-fucosidase protein levels in human crude extracts, combinding the slot-blot technique and a nonradioactive immunoassay. Taking advantage of the similarities in different mammalian fucosidases, a polyclonal antiserum was raised against commercial purified α-l-fucosidase from bovine kidney that cross-reacted with the human colon enzyme. The method is able to detect as little as 0.75 ng α-l-fucosidase. To illustrate the direct application of this technique, we analysed and quantified α-l-fucosidase protein levels in 18 human colon crude samples. This technique could prove useful in clinical pathology, allowing fast and accurate measurement of α-l-fucosidase in crude extracts.     

The identification of a new metabolite of phencyclidine

A new metabolite of phencyclidine, formed in incubation mixtures of rabbit liver preparations and phencyclidine, has been identified as N-(5-hydroxypentyl)-1-phenylcyclohexylamine (I). This compound could result from the reduction of an aldehyde generated by α-carbon hydroxylation of the piperidine ring of phencyclidine. The identification is based on comparison of gas chromatographic and mass spectral properties of metabolite with synthetic material.     

Detection of protein adduction derived from styrene oxide to cysteine residues by alkaline permethylation

Styrene oxide-cysteine adduction is predominantly involved in protein covalent modification after exposure in vivo to styrene or styrene oxide. In the present study, we developed an alkaline permethylation- and GC/MS-based approach to detect styrene oxide-derived protein adduction. Permethylation of the protein adducts produced two methylthiophenylethanols, namely 2-methylthio-2-phenyl-1-ethanol and 2-methylthio-1-phenyl-1-ethanol. To improve the permethylation efficiency, reaction conditions, including temperature, time, NaOH strength, and molar ratio of CH₃I/NaOH, were explored. Under optimized conditions, the yields of the analyte formation resulting from permethylation of authentic standard α- and β-mercapturic acids, representing α and β isomers of cysteine adducts, were 35% and 28%, respectively. Permethylation of styrene oxide-modified bovine serum albumin released the two methylthiophenylethanols with an α-/β-adduction ratio of 1.5. A concentration-dependent increase in both α- and β-adduction was observed in mouse liver microsomes incubated with styrene at various concentrations. CD-1 mice were administered intraperitoneally with styrene at doses of 0, 50, and 400 mg/kg daily for 5 days. The formation of protein adducts derived from styrene oxide in whole blood in 400 mg/kg group was observed with an α/β ratio of 4.8, suggesting that the reaction of styrene oxide with cysteine residues took place more likely at the α-carbon than the β-carbon of styrene oxide.     

Using molecular principal axes for structural comparison: determining the tertiary changes of a FAB antibody domain induced by antigenic binding

Background  

Comparison of different protein x-ray structures has previously been made in a number of different ways; for example, by visual examination, by differences in the locations of secondary structures, by explicit superposition of structural elements, e.g. α-carbon atom locations, or by procedures that utilize a common symmetry element or geometrical feature of the structures to be compared.