Hydrogen Exchange of Individual Amide Protons in the E. Coli lac Repressor DNA-binding Domain: A Nuclear Magnetic Resonance Study (29)

Abstract

Proton exchange in lac repressor headpiece was studied by COSY and 2D NOE spectroscopy. The exchange rates of amide protons, stabilized by the hydrogen bonds of the three α-helices of the headpiece, could be determined quantitatively. The exchange rates in these helices showed repetitive patterns of about three to four residues. A correlation with the position of the amide proton in the interior or the exterior of the α-helix of the protein was found. The exchange data strongly support the validity of the three-dimensional structure, as determined recently (Kaptein, R. et al., J. Mol. Biol. 182, 179-182 (1985)).     

Basis for large differences in the cooperativity of the formation of antiparallel β-sheets and clusters of interacting α-helices in isolated chains

Configuration partition functions that describe the intramolecular formation of antiparallel β-sheets and clusters of antiparallel interacting α-helices are very nearly of the same form. They can be interconverted by a simple change in notation and the addition of one weighting factor for each cluster of interacting α-helices. This extra weighting factor is the Zimm–Bragg σ which must be less than one. When it is assigned a reasonable numerical value, it plays an important role in the determination of the nature of the transition from the disordered chain to the ordered structure. It causes the formation of clusters of interacting α-helices to be more cooperative than the formation of antiparallel β-sheets in isolated chains.     

Energetics of multihelix interactions in protein folding: application to myoglobin

Conformational-energy calculations have been carried out in order to determine favorable packing arrangements within a group of α-helices. The influence of side chains and of the number of interacting α-helices on the mode of packing was analyzed. In this work, our earlier methods for computing the packing energy of a pair of α-helices [Chou, K.-C., Némethy, G. & Scheraga, H. A. (1984) J. Am. Chem. Soc. 106 , 3161–3170] have been extended to treat the interactions among several helices. Also, new algorithms allow the matching of standard peptide geometry to x-ray coordinates of helical complexes and the analysis of interrelations between several helices. As a specific test case, the packing of three neighboring α-helices, viz., the A, G, and H helices of sperm whale myoglobin, was considered. Minimum-energy arrangements were computed for the separate A-H and the G-H α-helix pairs as well as for the A-G-H three-helix complex. For the packing of the nearly antiparallel G and H α-helices, the same optimal structure was obtained in two- and three-helix complexes, indicating that a single packing arrangement is specifically favored by interhelix interactions. For the pair of nearly perpendicular A and H α-helices, interactions are less specific, so that there is no unique optimal structure in the two-helix complex; in the three-helix complex, however, a specific mode of packing is favored even for the A-H pair. This result indicates that the presence of other nearby α-helices can influence the packing of a given α-helix pair. The computed arrangement of the A-G-H complex is very close to that of the crystallographically determined structure. These results can be used to make deductions about the likely sequence of events in protein folding, where, in this particular case, it appears that the G-H helix pair may form first and then induce proper orientation of the A helix.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Biochemical and structural characterization of TLXI,the Triticum aestivum L. thaumatin-like xylanase inhibitor

Thaumatin-like xylanase inhibitors (TLXI) are recently discovered wheat proteins. They belong to the family of the thaumatin-like proteins and inhibit glycoside hydrolase family 11 endoxylanases commonly used in different cereal based (bio)technological processes. We here report on the biochemical characterisation of TLXI. Its inhibition activity is temperature- and pH-dependent and shows a maximum at approximately 40°C and pH 5.0. The TLXI structure model, generated with the crystal structure of thaumatin as template, shows the occurrence of five disulfide bridges and three β-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no α-helix is present. The circular dichroism spectrum of TLXI confirms the absence of α-helices and the presence of antiparallel β-sheets. All ten cysteine residues in TLXI are involved in disulfide bridges. TLXI is stable for at least 120 min between pH 1–12 and for at least 2 hours at 100°C, making it much more stable than the other two xylanase inhibitors from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP). This high stability can probably be ascribed to the high number of disulfide bridges, much as seen for other thaumatin-like proteins.     

Subunit Secondary Structure in Filamentous Viruses: Predictions and Observations

Abstract

The algorithm of Gamier, Osguthorpe and Robson (J. Mol. Biol. 120, 97–120, 1978) for prediction of protein secondary structure has been applied to the coat protein sequences of six filamentous bacteriophages: fd, Ifl, IKe, Pfl, Xf and Pf3. For subunits of Class I virions (fd, Ifl, IKe), the algorithm predicts a very high percentage of helix in comparison to other structure types, which is in accord with the results of laser Raman and circular dichroism measurements. For subunits of the Class II virions (Pfl, Xf, Pf3), the algorithm consistently predicts a predominance of β structure, which is compatible with the demonstrated facility for conversion of Class II subunits from α-helix to β-strand under appropriate experimental conditions (Thomas, Prescott and Day, J. Mol. Biol. 165, 321–356, 1983). Even when the algorithm is biased to favor helix, the Class II virion subunits are predicted to contain considerably more strand than helix. Qualitatively similar results are obtained using the algorithm of Chou and Fasman {Adv. Enzym. 47, 45–148,45-148). Therefore, both predictive and experimental methods indicate a distinction between Gass I and II subunits, which is reflected in a greater tendency of the latter to adopt other than uniform β-helical conformation. The results suggest a possible model for the disassembly of filamentous viruses which may involve the unraveling of coat protein helices at the N terminus.     

RNA-Protein Interactions in Some Small Plant Viruses

Abstract

The structure of the three quasi-equivalent protein subunits A, B and C of the spherical, T = 3 southern bean mosaic virus (SBMV) have been carefully built in accordance with a refined electron density map of the complete virus. The lower electron density in the RNA portion of the map could not be explicitly interpreted in terms of a preferred RNA structure on which some icosahedral symmetry might have been imposed. However, the extremely basic nature of the interior surface of the coat protein must be associated with the binding and organization of the RNA. Comparison with the small spherical, T = 1 satellite tobacco necrosis virus (STNV; Liljas et al., J. Mol. Biol. 159, 93–108,1982) and the T = 1 aggregate of alfalfa mosaic virus (AMV) protein (Fukuyama et al., J. Mol. Biol. 150, 33–41, 1981) showed similar results.

The pattern of basic residues on the SBMV coat protein surface facing the RNA is able to dock a 9 base pair double-helical A-RNA structure with surprising accuracy. The basic residues are each associated with a different phosphate and the protein can make interactions with five bases in the minor groove. This may be one of a small number of ways in which the RNA interacts with SBMV coat protein.

The self-assembly of SBMV has been studied in relation to the presence of the 63 basic amino-terminal coat protein sequence, pH, Ca²⁺ and Mg²⁺ ions and RNA. These results have led to a two-state model where the “relaxed” dimers initially self-assemble into 10-mer caps which nucleate the assembly of T = 1 or T = 3 capsids depending on the charge state of the carboxyl group clusters in the subunit contact region. The two-state condition of dimers in a viral coat protein extends the range of structures originally envisaged by Caspar and Klug (Cold Spring Harbor Symp. Quant. Biol. 27, 1–24, 1962).     

Circular dichroism of flow-oriented nucleic acids. II. Comparison between observed and calculated spectra.

The ultraviolet circular dichroism (CD) of oriented DNA and RNA molecules is calculated by an extension of Johnson and Tinoco's theory [(1969) Biopolymers 7 , 727–749] for unoriented molecules. The calculations are carried out for molecular models of A-DNA, B-DNA, planar B-DNA, C-DNA, and RNA-11-α. The calculated curves are compared to measured spectra [(1975) J. Mol. Biol. 92 , 449–466] Chung and Holzwarth, for oriented solutions of DNA in buffer, DNA in 6 M LiCl or in ethylene glycol, and double-stranded viral RNA. The calculation, which considers only base–base interactions, predicts that the CD of B-DNA, measured with light propagating parallel to the helix axis, should be large and semiconservative, whereas the CD for light propagating perpendicular to the helix axis should be nonconservative. These predictions agree qualitatively with the experimental observations for DNA in buffer; agreement improves if one assumes the bases to be exactly perpendicular to the helix axis. For the other geometries, agreement is less satisfactory, but qualitative agreement with experiment is obtained and the signs of the specific CD spectra are in accord with observations.     

Dynamics of the cluster model of protein folding

The cluster model of protein folding [Kanehisa, M. I. & Tsong, T. Y. (1978) J. Mol. Biol. 124 , 177–194] is further investigated for the thermodynamic and kinetic properties of protein folding–unfolding transitions. A cluster is a locally formed ordered region in the polypeptide chain due to cooperative interactions among residues. In the cluster model a cooperative term is assigned as proportional to the surface area of a globular cluster. This assignment is compared with that for the helix–coil transition of homopolypeptides, where the cooperative term is proportional to the two ends of a linear helical sequence. The dynamics of the cluster model exhibit a slow phase, which is well-separated from other faster phases, because of the cooperative interaction of the macrosystem. This slow phase not only appears within the transition region, but can also persist well below the transition region if the cooperativity depends on the external condition. The amplitudes of certain kinetic phases can vary depending on the choice of physical parameters monitoring the reaction. Thus the same reaction may display different time courses. The qualitative aspects of the folding dynamics are as follows. In one case the rate-limiting formation of a critical-size cluster is followed by its rapid growth, while in the other the rate-limiting step appears in a later stage, where preformed smaller clusters merge into larger ones. The former case is similar to the dynamics of the helix–coil transition, and the latter represents a stepwise mechanism of protein structure formation.     

Theoretical pi-pi absorption, circular dichroic, and linear dichroic spectra of collagen triple helices

Absorption, CD, and LD spectra of the π-π* transition near 200 nm are calculated for poly(Gly-X-Y) (X,Y = Gly, Ala, Pro) in four conformations proposed for collagen like triple helices in the recent literature. A dipole interaction model is used with the same optical paramenters as in previous studies of polypeptide spectra. The CD spectra are sensitive to backbone structure and amino acid composition, although the experimentally observed negative peak near 200 nm is a general feature of most the calculated spectra. Interchain interactions significantly affect the CD spectra in most cases. Calculations for (Gly-Pro-Ala)₃ and (Gly-Ala-Pro)₃ in the triple helical structure of Fraser, MacRae, and Suzuki [(1979) J. Mol. Biol. 129 , 463–481] show absorption, CD, and LD spectra in fairly good agreement with experiment. The characteristics of the π-π* normal modes responsible for the calculated spectra are compared with those of the component bands resolved from the experimental spectra of collagen by Mandel and Holzwarth [(1973) Biopolymers 12 , 655–674].     

Circular dichroism spectra show abundance of beta-sheet structure in connectin, a muscle elastic protein

Circular dichroism spectra of native connectin from chicken breast muscle strongly suggested the abundant presence of beta-sheet structure, as much as 70% in 0.5 M KCl and 50 mM phosphate buffer, pH 7.5. alpha-Helix was not detected. These results are in contradiction with the conclusion that native connectin from rabbit skeletal muscle consists entirely of random coil [(1984) J. Mol. Biol. 180, 331-356].     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Proton and phosphorus nmr investigation of the conformational states of acid polyadenylic double helix

Proton and phosphorus nmr have been used to investigate the double-helical structures of polyriboadenylic acid [poly(A)] formed in acidic solutions (pH < 6). The results obtained at low pH (～4.5) are consistent with the model for the acid poly(A) double helix proposed by Rich [Rich, A., Davies, D. R., Crick, F. H. C. & Watson, J. D. (1961) J. Mol. Biol. 3 , 71–86]. Other models that have been proposed are inconsistent with the nmr data. The nmr measurements have also been used to examine the conformation of poly(A) helix in the half-protonated state. Although the base-stacking arrangement of this state is similar to that observed in the more extensively protonated low-pH state, the phosphate backbone conformation is different from that found in either the neutral or low-pH structures.     

Insights into thermal resistance of proteins from the intrinsic stability of their α-helices

To investigate the role of α helices in protein thermostability, we compared energy characteristics of α helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family. The changes in intrinsic free energy of α-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices [Munoz, V., Serrano, L. Nature Struc. Biol. 1:399–409, 1994; Munoz, V., Serrano, L. J. Mol. Biol. 245:275–296, 1995; Munoz, V., Serrano, L. J. Mol. Biol. 245:297–308, 1995]. Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of α helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain–side chain interactions, are the main contributors to protein thermostability. Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of α helices is a general feature of many thermophilic proteins. The magnitude of the observed decrease in intrinsic free energy on α-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability. Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal. This indicates that certain flexibility of RecA protein structure is an essential factor for protein function. All RecA proteins analyzed fell into three temperature-dependent classes of similar α-helix stability (ΔG_int = 45.0 ± 2.0 kcal/mol). These classes were consistent with the natural origin of the proteins. Based on the sequences of protein α helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200°C. Proteins 29:309–320, 1997. © 1997 Wiley-Liss, Inc.     

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR study of molecular structure of the heme cavity

The molecular structure of the active site of myoglobin from the shark, Galeorhinus japonicus, has been studied by 1H-NMR. Some hyperfine-shifted amino acid proton resonances in the met-cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two-dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrE10 and IleFG5 residues relative to the heme in G. japonicus met-cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM (magnetic anisotropy tensor determination utilizing heme methyls) [Yamamoto, Y., Nanai, N. & Ch?j?, R. (1990) J. Chem. Soc., Chem. Commun., 1556-1557] and the results were compared with the crystal structure of P. catodon carbonmonoxy myoglobin [Hanson, J. C. & Schoenborn, B. P. (1981) J. Mol. Biol. 153, 117-124]. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.     

The conformation of eukaryotic cytochrome c around residues 39, 57, 59 and 74.

1H-n.m.r. studies of horse, tuna, Candida krusei and Saccharomyces cerevisiae cytochromes c showed that each of the proteins contains a similar cluster of residues at the bottom of the protein that assists in shielding the haem from the solvent. The relative positions of the residues forming these clusters vary continuously with temperature, and they change with the change in protein redox state. This conformational heterogeneity is discussed with reference to the conformational flexibility of cytochrome c around residues 57, 59 and 74. Spectroscopic measurements of pKa values for Lys-55 (horse and tuna cytochromes c) and His-33 and His-39 (C. krusei and S. cerevisiae cytochromes c) are in excellent agreement with expectations based on chemical-modification studies of horse cytochrome c. [Bosshard & Zürrer (1980) J. Biol. Chem. 255, 6694-6699] and on the X-ray-crystallographic structure of tuna cytochrome c [Takano & Dickerson (1981) J. Mol. Biol. 153, 79-94, 95-115].     

Relaxation kinetics of Helix pomatia alpha-hemocyanin. Whole-half molecule reaction at pH 5.7 in 0.4 M NaCl.

Two independent relaxation kinetics methods were used to study samples of α-hemocyanin kindly furnished to us by members of the Biochemical Laboratory of the University of Groningen. A Durrum-Gibson stopped-flow apparatus was used to obtain concentration-jump data in the light-scattering mode. A recently developed pressurejump light-scattering apparatus was used to obtain completely independent data. The studies were made in 0.1 m acetate buffer at pH 5.7 containing 0.4 m NaCl, conditions under which equilibrium light-scattering studies had been reported by Engelborghs and Lontie (1973, J. Mol. Biol., 77, 577–587). In the companion paper (Kegeles, 1977, Arch. Biochem. Biophys., 180, 530–536), a model is proposed, consisting of a system containing a mixture of reactive and unreactive whole molecules, from which data are derived for the formation constant of whole molecules from halves and the fraction of material which is capable of undergoing reaction. The present study uses this estimate of this fraction of reactive material to permit the evaluation of overall rate constants and equilibrium constants. When the estimate of 65% of reactive material derived without making nonideality corrections is applied to the kinetics data, very satisfactory agreement is obtained between the equilibrium constant acquired from equilibrium data and the equilibrium constants derived from the kinetics data.     

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

Site‐directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as ¹H^N is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean‐force potential (Smoluchowski equation); (iv) explicit‐atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the ¹H^N residue number and static magnetic field strength; the results are appreciably different from the Gillespie–Shortle model. At the same time, the Gillespie–Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between ¹H^N spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin‐labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme—using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates—lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random‐coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random‐coil protein in good solvent/poor solvent.