Tertiary Structure Level Research Articles

Straws in the Wind

1 Iwas surprised to find that 4 years has elapsed since I last wrote an editorial for the Journal of Alternative and Complementary Medicine (JACM) (Smith, 1999); I called it “Physicks and Physics.” My thesis then was that physics hardly enters into orthodox medicine or alternative and complementary medicine because biology does not have a theoretical and mathematical basis in physics. In this Guest Editorial, I shall look for straws blowing in the wind of change since then. This time, my thesis is that “environmental medicine” and “alternative and complementary medicine” need each other. Although chemistry is readily comprehended in terms of balls-on-sticks, what is lost is that duality between electromagnetic frequency and the chemical bond that makes spectroscopic analysis possible. Chemistry, similar to spectroscopy, is based in quantum physics, and mathematical chemists can do sophisticated calculations about molecules and their structures. The theoretical basis for chemical changes brought about in living systems by electromagnetic fields is still an open question. One windblown straw is that the microwave cooker frequency (2.45 GHz) can trigger the isomeric transition of L-amino acids to D-amino acids (Lubec et al., 1989). Most such effects are biphasic and there will be another frequency that effects the reverse transition. This suggests that frequency effects can occur at least at the level of tertiary molecular structure and involve enzyme activity. In the myth of Achilles and the Tortoise, the choice of the wrong methodology ensures that the instant at which Achilles overtakes the Tortoise is never reached this side of infinity. It seems to me that this describes many aspects of alternative and complementary medicine whereby the wrong methodology is attempted or enforced by critics. An example of this is the ongoing controversy concerning possible health risks associated with the electromagnetic environment: mobile phones, transmitters, power lines, and so on. Official sources and institutions tell us that there are no health problems until power levels are such that thermal effects set in, the “English muffin syndrome”—”If it’s not burned it’s alright!” This is based on the methodology of classic (prequantum) physics within which there is no chemistry or spectroscopy. My view is that problems involving the electromagnetic environment are only explicable in terms of an alternative medicine that recognizes that there are those pathways to the body’s regulatory systems that are accessed through acupuncture, acupressure, electroacupuncture, homeopathy, and so on, as well as through mind–body interactions. A study called, “Cancer Incidence near Radio and Television Transmitters in Great Britain II. All High Power Transmitters” (Dolk et al., 1997) concluded that, “. . . . while there is evidence of a decline in leukaemia risk with distance from transmitters, the pattern and

HIV-1 RNA Dimerization Initiation Site Is Structurally Similar to the Ribosomal A Site and Binds Aminoglycoside Antibiotics

Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.

Impact of domain interchange on conformational stability and equilibrium folding of chimeric class micro glutathione transferases.

Rat micro class glutathione transferases M1-1 and M2-2 are homodimers that share a 78% sequence identity but display differences in stability. M1-1 is more stable at the secondary and tertiary structural levels, whereas its quaternary structure is less stable. Each subunit in these proteins consists of two structurally distinct domains with intersubunit contacts occurring between domain 1 of one subunit and domain 2 of the other subunit. The chimeric subunit variants M(12), which has domain 1 of M1 and domain 2 of M2, and its complement M(21), were used to investigate the conformational stability of the chimeric homodimers M(12)-(12) and M(21)-(21) to determine the contribution of each domain toward stability. Exchanging entire domains between class micro GSTs is accommodated by the GST fold. Urea-induced equilibrium unfolding data indicate that whereas the class micro equilibrium unfolding mechanism (i.e., N(2) <--> 2I <--> 2U) is not altered, domain exchanges impact significantly on the conformational stability of the native dimers and monomeric folding intermediates. Data for the wild-type and chimeric proteins indicate that the order of stability for the native dimer (N(2)) is M2-2 > M(12)-(12) M1-1 approximately M(21)-(21), and that the order of stability of the monomeric intermediate (I) is M1 > M2 approximately M(12) > M(21). Interactions involving Arg 77, which is topologically conserved in GSTs, appear to play an important role in the stability of both the native dimeric and folding monomeric structures.

Creating nanocavities of tunable sizes: hollow helices.

A general strategy for creating nanocavities with tunable sizes based on the folding of unnatural oligomers is presented. The backbones of these oligomers are rigidified by localized, three-center intramolecular hydrogen bonds, which lead to well-defined hollow helical conformations. Changing the curvature of the oligomer backbone leads to the adjustment of the interior cavity size. Helices with interior cavities of 10 A to >30 A across, the largest thus far formed by the folding of unnatural foldamers, are generated. Cavities of these sizes are usually seen at the tertiary and quaternary structural levels of proteins. The ability to tune molecular dimensions without altering the underlying topology is seen in few natural and unnatural foldamer systems.

The use of protein structure/activity relationships in the rational design of stable particulate delivery systems.

The recombinant heat shock protein (18 kDa-hsp) from Mycobacterium leprae was studied as a T-epitope model for vaccine development. We present a structural analysis of the stability of recombinant 18 kDa-hsp during different processing steps. Circular dichroism and ELISA were used to monitor protein structure after thermal stress, lyophilization and chemical modification. We observed that the 18 kDa-hsp is extremely resistant to a wide range of temperatures (60% of activity is retained at 80 degrees C for 20 min). N-Acylation increased its ordered structure by 4% and decreased its beta-T1 structure by 2%. ELISA demonstrated that the native conformation of the 18 kDa-hsp was preserved after hydrophobic modification by acylation. The recombinant 18 kDa-hsp resists to a wide range of temperatures and chemical modifications without loss of its main characteristic, which is to be a source of T epitopes. This resistance is probably directly related to its lack of organization at the level of tertiary and secondary structures.

Structure determination of human and murine beta-defensins reveals structural conservation in the absence of significant sequence similarity.

Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian beta-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human beta-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

Hierarchical Structure−Ferroelectricity Relationships of Barium Titanate Particles

The aim of this paper is to dissect the hierarchical structures of BaTiO3 particles into primary, secondary, tertiary, and quaternary structural levels and to thoroughly review the corresponding structure−ferroelectricity relationships at all levels. This analytical framework is of growing importance for the fundamental understanding of the ferroelectric properties of BaTiO3 in microelectronic devices, especially as their structural levels are all approaching the same scale in the process of miniaturization. The identification of the most influential structure among others and the deduction of a meaningful structure−ferroelectricity relationship depend on a thorough understanding of the relationships.

Simulation of protein folding by reaction path annealing

We present a systematic application of reaction path sampling to computer simulations of the folding of peptides and small proteins at atomic resolution in the presence of solvent. We use a simulated annealing protocol to generate an ensemble of room temperature folding trajectories of fixed length, which connect predetermined initial and final states. The trajectories are distributed according to a discretized version of the Onsager–Machlup action functional. We show that, despite the enormous practical restrictions placed on the number of time slices which can be explored, some of the basic kinetic features found experimentally for the folding of peptides and small proteins are exhibited in the nature of the reaction paths sampled. We test the method on three systems: A 12 residue α-helical peptide, a 16 residue β-hairpin peptide, and the 36 residue avian Pancreatic Polypeptide (aPP). All systems are represented at atomic resolution, and include explicit water molecules. For the 12 residue α-helix, we find that (i,i+3) hydrogen bonds can play a significant role in the folding pathway, with specific (i,i+3) bonds appearing, then transforming to the corresponding (i,i+4) hydrogen bond for some, but not all of the native hydrogen bonds. For the β-hairpin and aPP, hydrophobic interactions play a dominant role, with nonbonded interactions consistently appearing before hydrogen bonds. This is true both at the level of tertiary structure, and at the level of individual hydrogen bonds which tend to form only after stabilizing nonbonded interactions have already formed between the residues involved.

Sequence-structure specificity of a knowledge based energy function at the secondary structure level.

This paper investigates the sequence-structure specificity of a representative knowledge based energy function by applying it to threading at the level of secondary structures of proteins. Assessing the strengths and weaknesses of an energy function at this fundamental level provides more detailed and insightful information than at the tertiary structure level and the results obtained can be useful in tertiary level threading. We threaded each of the 293 non-redundant proteins onto the secondary structures contained in its respective native protein (host template). We also used 68 pairs of proteins with similar folds and low sequence identity. For each pair, we threaded the sequence of one protein onto the secondary structures of the other protein. The discerning power of the total energy function and its one-body, pairwise, and mutation components is studied. We then applied our energy function to a recent study which demonstrated how a designed 11-amino acid sequence can replace distinct segments (one segment is an alpha-helix, the other is a beta-sheet) of a protein without changing its fold. We conducted random mutations of the designed sequence to determine the patterns for favorable mutations. We also studied the sequence-structure specificity at the boundaries of a secondary structure. Finally, we demonstrated how to speed up tertiary level threading by filtering out alignments found to be energetically unfavorable during the secondary structure threading. The program is available on request from the authors. xud@ornl.gov

Opposite Effects of Ca2+ and GTP Binding on Tissue Transglutaminase Tertiary Structure

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.

The single mutation Phe173 --> Ala induces a molten globule-like state in murine interleukin-6.

A series of three aromatic to alanine mutants of recombinant murine interleukin-6 lacking the 22 N-terminal residues (DeltaN22mIL-6) were constructed to investigate the role of these residues in the structure and function of mIL-6. While Y78A and Y97A have activities similar to that of DeltaN22mIL-6, F173A lacks biological activity. F173A retains high levels of secondary structure, as determined by far-UV circular dichroism (CD), but has substantially reduced levels of tertiary structure, as determined by near-UV CD and (1)H NMR spectroscopy. F173A also binds the hydrophobic dye 1-anilino-8-naphthalenesulfonic acid (ANS) over a range of pH values and exhibits noncooperative equilibrium unfolding (as judged by the noncoincidence of monophasic unfolding transitions monitored by far-UV CD and lambda(max), with midpoints of unfolding at 2.6 +/- 0. 1 and 3.5 +/- 0.3 M urea, respectively, and the lack of an observable thermal unfolding transition). These are all properties of molten globule states, suggesting that the loss of activity of F173A results from the disruption of the fine structure of the protein, rather than from the loss of a side chain that is important for ligand-receptor interactions. Surprisingly, under some conditions, this loosened conformation is no more susceptible to proteolytic attack than the parent protein. By analogy with human IL-6, Phe173 in DeltaN22mIL-6 makes multiple interhelical interactions, the removal of which appear to be sufficient to induce a molten globule-like conformation.

Physicochemical characterization of an antagonistic human interleukin-6 dimer.

A noncovalently bound dimeric form of recombinant human IL-6 interleukin-6 (IL-6D) was shown to be an antagonist for IL-6 activity, in a STAT3 tyrosine phosphorylation assay using HepG2 cells, under conditions where it does not dissociate into monomeric IL-6 (IL-6M). The fluorescence from Trp157, the single tryptophan residue in the primary sequence of IL-6, is altered in IL-6D, where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. These data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6D and IL-6M are compact molecules, as determined by sedimentation velocity analysis, and contain essentially identical levels of secondary and tertiary structure, as determined by far- and near-UV CD, respectively. IL-6D and IL-6M show the same susceptibility to limited proteolytic attack, and exhibit identical far-UV CD-monitored urea-denaturation profiles with the midpoint of denaturation occurring at 6.0 +/- 0.1 M urea. However, IL-6D was found to dissociate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimerization occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6D is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from each protein chain are swapped through the loop regions at the "top" of the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonistic activity of IL-6D. In this model, receptor binding site I, which comprises residues in the A/B loop and the C-terminus of the protein, is free to bind the IL-6 receptor. However, site III, which includes Trp157 and residues in the C/D loop and N-terminal end of helix D, and perhaps site II, which comprises residues in the A and C helices, are no longer able to bind the signal transducing component of the IL-6 receptor complex, gp130.

The Analgesic Domain of IL-2

Human recombinant interleukin-2 (IL-2) has been showed to exert an analgesic effect and some experiments suggested that opioid receptors were involved. Because the aromatic residues at the N-termini of opioid peptides are crucial for their binding to the opioid receptors, all nine aromatic residues in IL-2 were mutated by site-directed mutagenesis and both the analgesic activity and the immune activity of the wild-type and mutated IL-2 were tested. The result indicated that there exists a putative analgesic function domain in IL-2, in which Phe44, Tyr45, Tyr107, and Phe117 were essential. These four residues are located close together at the tertiary structure level of IL-2.

Thermal unfolding of the cytotoxin α-sarcin: phospholipid binding induces destabilization of the protein structure

The effect of membrane binding on the structure and stability of the cytotoxin α-sarcin has been studied by differential scanning calorimetry, Fourier-transform infrared and fluorescence spectroscopic techniques. The thermal unfolding of α-sarcin in aqueous solution fits into a two-state transition characterized by a transition temperature ( T m) of 52.6°C and a calorimetric enthalpy ( ΔH cal) of 136 kcal/mol. Upon interaction with phosphatidylglycerol vesicles, α-sarcin undergoes conformational changes, as deduced from the FTIR and fluorescence emission spectra. These changes result in a decreased T m and ΔH cal values for the thermal unfolding of phospholipid-bound α-sarcin. The lower T m value for lipid-bound α-sarcin is also observed at the level of secondary and tertiary structures, based on analyses of both the amide I′ infrared spectrum and the tryptophan emission of the protein as a function of temperature, respectively. The results obtained indicate a protein destabilization promoted by the phospholipid interaction.

Sequence and Structure Requirements for Drosophila tRNA 5′- and 3′-End Processing

Eukaryotic tRNAs are processed at their 5'- and 3'- ends by endonucleases RNase P and 3'-tRNase, respectively. We have prepared substrates for both enzymes, separated the activities from a Drosophila extract, and designed variant tRNAs to assess the effects of sequence and structure on processing. Mutations affect these reactions in similar ways; thus, RNase P and 3'-tRNase probably require similar substrate structures to maintain the catalytic fit. RNase P is more sensitive to substrate substitutions than 3'-tRNase. In three of the four stems, one substitution prevents both processing reactions while the opposite one has less effect; anticodon stem substitutions hardly affect processing, and double substitution intended to restore base pairing also restore processing to the wild type rate. Structure probing suggests that tRNA misfolding sometimes coincides with reduced processing. In other cases, processing inhibition probably results from specific unfavorable stem appositions leading to local helix deformation. A single T loop substitution disrupts the tertiary D-T loop interaction and reduces processing. We have thus begun mapping tRNA processing determinants on the global, local, and tertiary structure levels.

Protein structure prediction: recognition of primary, secondary, and tertiary structural features from amino acid sequence.

This review attempts a critical stock-taking of the current state of the science aimed at predicting structural features of proteins from their amino acid sequences. At the primary structure level, methods are considered for detection of remotely related sequences and for recognizing amino acid patterns to predict posttranslational modifications and binding sites. The techniques involving secondary structural features include prediction of secondary structure, membrane-spanning regions, and secondary structural class. At the tertiary structural level, methods for threading a sequence into a mainchain fold, homology modeling and assigning sequences to protein families with similar folds are discussed. A literature analysis suggests that, to date, threading techniques are not able to show their superiority over sequence pattern recognition methods. Recent progress in the state of ab initio structure calculation is reviewed in detail. The analysis shows that many structural features can be predicted from the amino acid sequence much better than just a few years ago and with attendant utility in experimental research. Best prediction can be achieved for new protein sequences that can be assigned to well-studied protein families. For single sequences without homologues, the folding problem has not yet been solved.

The structure of DNA junctions and their interaction with enzymes.

Branched DNA species are commonly postulated as intermediates in DNA rearrangements, such as genetic recombination. The four-way junction was proposed to be the central intermediate in homologous genetic recombination [1—7] and there is good evidence for a role in the integrase class of site- specific recombination events [8—11]. Branched DNA can also arise during replication of DNA, such as the replication of T4 DNA. In each case, enzymes are required to interact specifically with these structures, in order to bring about a resolution or repair event. Such proteins achieve impressive feats of moelcular recognition at the level of DNA tertiary structure, providing us with a fascinating challenge to understand how this is brought about. However, it has been our belief that the key to this is first to analyse the structure of the DNA substrate, the four-way DNA junction. What follows is a description of our current view of the structure of the four- way DNA junction, and the way this is recognised by proteins. Inevitably, this is seen from the somewhat personal perspective of ideas and results from our own laboratories.

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such ‘knowledge’ in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential ‘tools’ for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.

A spectroscopic study of the conformational properties of foamed bovine serum albumin

The spectral properties of resolubilized foamed bovine serum albumin (BSA) have been compared with native BSA and the results interpreted in terms of the conformational properties of the proteins. Far-UV circular dichroism spectra reveal only minor changes in the protein secondary structure evidenced by a small reduction in helix content after foaming, although this may be significant. The biggest differences in conformation appear to be at the tertiary structural level and are readily detected by intrinsic fluorescence. A major, irreversible reduction (>30%) in the intensity of the tryptophan emission is reproducibly observed in the foamed sample. In addition, the emission spectrum of the foamed sample exhibits an ionic strength dependence which is not observed in native BSA. Quenching studies with iodide over a range of ionic strength reveal that the properties of both tryptophans in the foamed sample are affected as they both behave in a different manner from those of the native protein. The change in conformation induced by foaming does not apparently reflect a change in the state of aggregation of the foamed protein. The native and foamed BSA samples used in these experiments contain similar amounts of oligomer as judged by nondenaturing polyacrylamide gel electrophoresis. These data have been used to gain a clearer understanding of the nature of the irreversible conformational change resulting from adsorption of BSA at the air/water interface of a foam.

Characterization of the rat retinol-binding protein gene and its comparison to the three-dimensional structure of the protein.

Rat genomic DNA fragments bearing the retinol-binding protein (RBP) gene have been isolated and characterized. The gene spans 6.9 kilobases and contains six exons. The five intervening sequences range in size from 78 base pairs to 4.4 kilobase pairs with the first interrupting the 5' untranslated region. A comparison of the gene organization with the three-dimensional structure of RBP reveals that all translated exon transcripts closely correspond to discrete tertiary structural elements. Residues of the protein involved in the retinol binding are encoded by three separate exons. It has been proposed that the two regions displaying internal homology in the human RBP, both at the primary and tertiary structure levels, arose by a partial ancestral gene duplication. If such an event were involved, evidence for it at the nucleotide sequence and exon-intron organization levels has been obscured.