Monomer-dimer Transition Research Articles

A Fraction of the BglG Transcriptional Antiterminator from Escherichia coli Exists as a Compact Monomer

Expression of the bgl operon in Escherichia coli, induced by beta-glucosides, is positively regulated by BglG, a transcriptional antiterminator. In the presence of inducer, BglG dimerizes and binds to the bgl transcript to prevent premature termination of transcription. The dimeric state of BglG is determined by BglF, a membrane-bound enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which reversibly phosphorylates BglG according to beta-glucoside availability. BglG is composed of an RNA-binding domain followed by two homologous PTS regulation domains (PRD1 and PRD2). The predicted structure of dimeric LicT, a BglG homologue from Bacillus subtilis, suggests that the two PRDs adopt a similar structure and that the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have shown recently that the PRD1 and PRD2 domains of BglG can form a stable heterodimer. We report here, based on in vitro and in vivo cross-linking experiments, that a fraction of BglG is present in the cell in a compact form in which PRD1 and PRD2 are in close proximity. The compact form is present mainly in the BglG monomers. Our results imply that the monomer-dimer transition involves a conformational change. The possible role of the compact form in preventing untimely induction of the bgl operon is discussed.

Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions.

Dimerization is proposed to be a regulatory mechanism for TATA-binding protein (TBP) activity both in vitro and in vivo. The reversible dimer-monomer transition of TBP is influenced by the buffer conditions in vitro. Using in vitro chemical cross-linking, we found yeast TBP (yTBP) to be largely monomeric in the presence of the divalent cation Mg2+, even at high salt concentrations. Apparent molecular mass of yTBP at high salt with Mg2+, run through a gel filtration column, was close to that of monomeric yTBP. Lowering the monovalent ionic concentration in the absence of Mg2+, resulted in dimerization of TBP. Effect of Mg2+ was seen at two different levels: at higher TBP concentrations, it suppressed the TBP dimerization and at lower TBP levels, it helped keep TBP monomers in active conformation (competent for binding TATA box), resulting in enhanced TBP-TATA complex formation in the presence of increasing Mg2+. At both the levels, activity of the full-length TBP in the presence of Mg2+ was like that reported for the truncated C-terminal domain of TBP from which the N-terminus is removed. Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar mechanism.

Effect of high shear rate on stability of proteins: kinetic study

Size-exclusion chromatography (SEC) was used to monitor the time–course of protein degradation induced by high shear rates during the formulation and manufacture of controlled-release pharmaceutical dosage forms. SEC with multi-angle laser light-scattering (MALLS) detection was used to characterize the aggregation products, determining their absolute molecular weight. A stability-indicating method was developed and validated to obtain reliable drug degradation data. The results obtained according to the ICH guidelines confirm that the system and methods proposed are suitable for their intended use. The degradation kinetics are influenced by the type of protein and the effect of the shear rate on their stability. Reversible pseudo-first order degradation kinetics were observed for bovine β-lactoglobulin, whereas for human (HSA) and bovine serum albumin (BSA), a monomer–dimer transition was observed, independently of the rate of shear. However, trimer formation was also observed for HSA, especially at high shear rates. The kinetic model may thus be described as a two-step process: a monomer–dimer, and dimer–trimer transition.

Recognition of the Intrinsically Flexible Addiction Antidote MazE by a Dromedary Single Domain Antibody Fragment: STRUCTURE, THERMODYNAMICS OF BINDING, STABILITY, AND INFLUENCE ON INTERACTIONS WITH DNA

The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions. It encodes the toxic and long-lived MazF and the labile antidote MazE. The denaturation of MazE is a two-state reversible dimer-monomer transition. At lower concentrations the denatured state is significantly populated. This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell. Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques. Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation. The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e. three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions. A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data. Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.

Active-site Copper and Zinc Ions Modulate the Quaternary Structure of Prokaryotic Cu,Zn Superoxide Dismutase

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15–20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer–monomer transition. The dimer dissociation process is still incomplete at 6.5kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, ΔV, and the dissociation constant Kd0, at atmospheric pressure, (25ml/mol and 3×10−7M, respectively). The holoprotein is fully dimeric even at 6.5kbar, which allows us to evaluate a lower ΔG° limit of 11.5kcal/mol, corresponding to a dissociation constant Kd0<10−9M.

Numerical Study of the Entropy Loss of Dimerization and the Folding Thermodynamics of the GCN4 Leucine Zipper

A lattice-based model of a protein and the Monte Carlo simulation method are used to calculate the entropy loss of dimerization of the GCN4 leucine zipper. In the representation used, a protein is a sequence of interaction centers arranged on a cubic lattice, with effective interaction potentials that are both of physical and statistical nature. The Monte Carlo simulation method is then used to sample the partition functions of both the monomer and dimer forms as a function of temperature. A method is described to estimate the entropy loss upon dimerization, a quantity that enters the free energy difference between monomer and dimer, and the corresponding dimerization reaction constant. As expected, but contrary to previous numerical studies, we find that the entropy loss of dimerization is a strong function of energy (or temperature), except in the limit of large energies in which the motion of the two dimer chains becomes largely uncorrelated. At the monomer-dimer transition temperature we find that the entropy loss of dimerization is approximately five times smaller than the value that would result from ideal gas statistics, a result that is qualitatively consistent with a recent experimental determination of the entropy loss of dimerization of a synthetic peptide that also forms a two-stranded α-helical coiled coil.

Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK 27GFP and TK 366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK 366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 ± 1.6°, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 Å. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK 366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

Accessing the global minimum conformation of stefin A dimer by annealing under partially denaturing conditions

Stefin A folds as a monomer under strongly native conditions. We have observed that under partially denaturing conditions in the temperature range from 74 to 93 °C it folds into a dimer, while it is monomeric above the melting temperature of 95 °C. Below 74 °C the dimer is trapped and it does not dissociate. The dimer is a folded and structured protein as judged by CD and NMR, nevertheless it is no more functional as an inhibitor of cysteine proteases. The monomer-dimer transition proceeds at a slow rate and the activation energy of dimerization at 99 kcal/mol is comparable to the unfolding enthalpy. A large and negative dimerization enthalpy of −111(±8) kcal/mol was calculated from the temperature dependence of the dissociation constant. An irreversible pretransition at 10–15 deg. below the global unfolding temperature has been observed previously by DSC and can now be assigned to the monomer-dimer transition. Backbone resonances of all the dimer residues were assigned using 15N isotopically enriched protein. The dimer is symmetric and the chemical shift differences between the monomer and dimer are localized around the tripartite hydrophobic wedge, which otherwise interacts with cysteine proteases. Hydrogen exchange protection factors of the residues affected by dimer formation are higher in the dimer than in the monomer. The monomer to dimer transition is accompanied by a rapid exchange of all of the amide protons which are protected in the dimer, indicating that the transition state is unfolded to a large extent. Our results demonstrate that the native monomeric state of stefin A is actually metastable but is favored by the kinetics of folding. The substantial energy barrier which separates the monomer from the more stable dimer traps each state under native conditions.

ToxR co-operative interactions are not modulated by environmental conditions or periplasmic domain conformation.

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using lambda repressor-ToxR chimeric proteins and a lambda repressor-controlled reporter system (OR1 PR-lacZY) established that ToxR sequences can effectively dimerize the amino-terminal domain of lambda repressor in Escherichia coli. However, in E. coli, ToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of lambda-ToxR chimeras. When the OR1 PR-lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that lambda-ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of lambda-ToxR proteins to form tetramers in E. coli suggested that lambda-ToxR dimers could act co-operatively. Using a redesigned reporter system containing multiple lambda operator sites (OR1 OR2 OR3 PR-lacZY), we found that lambda-ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co-operative interactions. However, we falled to correlate a monomer-dimer transition of the lambda-ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re-evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. cholerae, deletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild-type levels of toxin in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.

Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) -- evidence of dimer-monomer equilibria.

Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-1), and 1-115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size. SDS/PAGE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS-2 was almost entirely dimeric at > 4 microM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.

Independence of Two Conformations of Sarcoplasmic Reticulum Ca2+-ATPase Molecules in Hydrolyzing Acetyl Phosphate: A TWO-PAIR MODEL OF THE ATPase STRUCTURAL UNIT

The sarcoplasmic reticulum Ca2+-ATPase molecules have been shown to exist in two conformations (A and B) that result from intermolecular interaction of ATPase molecules (Nakamura, J., and Tajima, G. (1995) J. Biol. Chem. 270, 17350-17354). The A form binds two calcium ions noncooperatively, whereas the B form binds the calcium ions cooperatively. Here, we examined the independence of these two forms in the calcium-activated hydrolysis of acetyl phosphate (AcP) under asynchronous and synchronous conditions of their E1-E2 transitions at 0-5 and 25 degrees C. Irrespective of their synchronism and temperature, the two forms hydrolyzed AcP due to calcium that was bound to each of the forms, indicating the independence of the two forms in hydrolyzing AcP. Taking into account the monomer-dimer transition of the ATPase molecules on the sarcoplasmic reticulum membrane accompanying E1-E2 transition of the molecules (Dux, L., Taylor, K. A., Ting-Beall, H. P., and Martonosi, A. (1985) J. Biol. Chem. 260, 11730-11743), the two types of molecules seem to independently carry out such monomer-dimer transition of the same type of molecules. Two pairs, each consisting of the same type of molecules, are suggested to be the structural unit of the ATPase molecules.

Glycinamide Ribonucleotide Transformylase Undergoes pH-dependent Dimerization

Glycinamide ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5). We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state. In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering. The data give a best fit to a cooperative three-proton transfer mechanism: 2M + 3H +⇌MH 2 2++ MH +⇌DH 3 3+ A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization. Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization. Fitting of the normalized titration curves yields an apparent p K a=7.16(±0.02) and a subnanomolar K Dfor the transition. Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization. There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization. Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution. Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell.

Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase.

The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism, and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M GuCl, gives rise to a dimeric inactive species which has lost pyridoxal 5'-phosphate (PLP), and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl, involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC, appears to be complete at 4.5 M GuCl, as indicated by the lack of further substantial changes in any of the signals being studied. Attempts at refolding resulted in the findings that: (1) partial reactivation is observed only starting from enzyme denatured at concentrations below 1.5 M GuCl, and (2) starting from completely denatured protein, the refolding process is apparently reversible down to concentrations of approx. 2 M GuCl. Taken together, this would seem to indicate that the monomer-dimer transition is impaired under the experimental conditions tested. A plausible model is presented for the unfolding/refolding of pkDDC.

Extraordinary Monomer-Dimer Transition of Methylene Blue Induced by the Phase Transition of Telomer-Bilayer Membranes Formed from Dialkyl L-Glutamate Amphiphiles with Oligo-Acrylic Acid-Head Group

Abstract Novel anionic telomer-bilayers formed from dialkyl L-glutamates with oligo-acrylic acid-head group were prepared. Methylene Blue interacted with these bilayers to form both monomeric and dimeric species. These monomeric species were converted to the dimeric species due to the gel-to-liquid crystalline phase transition of bilayer matrix. A similar phenomenon was observed even in the case of Acridine Orange.

ATPase activity of the UvrA and UvrAB protein complexes of theEscherichia coliUvrABC endonuclease

We have analyzed the ATPase activity exhibited by the UvrABC DNA repair complex. The UvrA protein is an ATPase whose lack of DNA dependence may be related to the ATP induced monomer-dimer transitions. ATP induced dimerization may be responsible for the enhanced DNA binding activity observed in the presence of ATP. Although the UvrA ATPase is not stimulated by dsDNA, such DNA can modulate the UvrA ATPase activity by decreases in Km and Vm and alterations in the Ki for ADP and ATP-gamma-S. The induction of such changes upon binding to DNA may be necessary for cooperative interactions of UvrA with UvrB that result in a DNA stimulated ATPase for the UvrAB protein complex. The UvrAB ATPase displays unique kinetic profiles that are dependent on the structure of the DNA effector. These kinetic changes correlate with changes in footprinting patterns, the stabilization of protein complexes on DNA damage and with the expression of helicase activity.

Proteolytic dimers of porcine muscle lactate dehydrogenase: characterization, folding, and reconstitution of the truncated and nicked polypeptide chain.

Lactate dehydrogenase from porcine skeletal muscle is a "dimer of dimers" that is stabilized in its tetrameric state by an N-terminal "arm" of approximately 20 amino acid residues. Due to the low dissociation constant of the tetramer, the dimer is inaccessible to direct analysis. Limited proteolysis during reconstitution (after dissociation at pH 2.3) yields stable "dimers". As suggested by affinity chromatography, these inactive dimers contain the dinucleotide fold of native LDH. In the presence of structure-making ions, approximately 40% activity is restored in the dimeric state [Girg, R., Jaenicke, R., & Rudolph, R. (1983) Biochem. Int. 7, 443-444]. The cleavage yields about equal amounts of three fragments, F 34, F 21, and F 14 (Mr 33.5K, 21.4K, and 13.5K, respectively). F 34 represents the intact chain lacking the N-terminal 10-11 amino acid residues; its C-terminus is heterogeneous, varying in the range between residues 326 +/- 5. F 21 contains residues 11/12 to 200 +/- 3; F 14 is a mixture of three subfragments: residues 11/12 to approximately 133, 38 to approximately 163, and 208 to approximately 327. After solubilization in 6 M guanidine hydrochloride, F 34 can be reconstituted to partially active dimers. Reactivation is determined by slow subunit refolding with subsequent diffusion-controlled dimerization, in accordance with the monomer-dimer transition in the reconstitution mechanism of the intact tetramer. Reconstitution of F 21 and F 14 is concentration dependent and leads to partially active "nicked dimers", indicating that separate domains are able to reassociate correctly to yield the native subunit arrangement.

Redetermination of the Crystal Structure and the Electrical Resistivity of Rb–TCNQ-II

Abstract The crystal structure of Rb–TCNQ-II was redetermined. The interplanar distance between the neighbouring TCNQ’s is 3.25 Å, which is almost equal to those of highly conducting TCNQ salts. The electrical resistivity and the structure of the TCNQ, columns suggest that the complete charge transfer in Rb–TCNQ-II makes this crystal low-conducting. The activation energy of the electrical conduction indicates that the dimerization gap appears below the monomer-dimer transition temperature. The temperature dependence of the electrical resistivities of Rb2TCNQ3 is also described.

On the 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. Purification and properties.

The steroid‐induced 3α‐hydroxysteroid:NAD+‐oxidoreductase from Pseudomonas testosteroni (ATCC 11996) exhibits reversible, concentration‐dependent monomer‐dimer transitions. The dimerization is demonstrable by gel chromatography and by precipitations with ammonium sulphate in concentrated solutions of the enzyme. The dimer formation leads to a considerable decrease in the specific activity, especially when androsterone is used as substrate. In this case the formation of dimers results in a 90 % reduction of the specific activity found with the monomers. The described effect is demonstrable, but less pronounced, when tetrahydrocortisone or deoxycholate are the substrates.Higly purified preparations of the 3α‐hydroxysteroid dehydrogenase prepared by repeated isoelectric focusings at different pH gradients have shown the enzyme to be composed of at least two isoenzymes. One major part focusing at pH 6.2 and a minor part focusing at pH 5.8.

Various Aspects of the Crystal Structures of the Molecular Compounds of TCNQ

Emphasis has been placed to the phenomena which seem to clearly show the nature of the one-dimensional array of TCNQ and to the X-ray analyses of two types of the superlattice structures of a molecular complex and an anion radical salt of TCNQ. The subjects outlined are as follows: (1) the possibility of a neutral↔ionic transition of the ground state charge distribution in the mixed-stacking columns of charge transfer complexes, (2) the relation between electrical conductivities and columnar structures of TCNQ anion radical salts, (3) Peierls instability of one-dimensional metal, (4) charge distribution and localization of excess electrons in TCNQ columns, (5) the evidence for monomer-dimer transition of alkali metal-TCNQ from X-ray diffraction patterns of these salts, (6) observation of X-ray satellite reflexion and the analysis of the sinusoidal structure of PTZ-TCNQ, (7) X-ray analysis of the anti-phase domain structure and stacking disorder of NH4-TCNQ.

Spectral peculiarities of the monomer-dimer transition of the phospholipases A 2 of Crotalus adamanteus venom.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSpectral peculiarities of the monomer-dimer transition of the phospholipases A2 of Crotalus adamanteus venomMichael A. WellsCite this: Biochemistry 1971, 10, 22, 4078–4083Publication Date (Print):October 1, 1971Publication History Published online1 May 2002Published inissue 1 October 1971https://doi.org/10.1021/bi00798a011RIGHTS & PERMISSIONSArticle Views29Altmetric-Citations13LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (629 KB) Get e-Alerts Get e-Alerts