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1.
Mizuguchi M Matsuura A Nabeshima Y Masaki K Watanabe M Aizawa T Demura M Nitta K Mori Y Shinoda H Kawano K 《Proteins》2005,61(2):356-365
The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin. 相似文献
2.
Lambda cro repressor complex with OR3 operator DNA. 19F nuclear magnetic resonance observations 总被引:4,自引:0,他引:4
The interaction of lambda cro repressor with DNA is probed using synthetic 17 base-pair OR3 operators in which 5-fluorodeoxyuridine has been systematically incorporated at each of the nine positions normally occupied by a thymidine residue. By monitoring changes in chemical shift of the fluorine resonances upon cro repressor binding in aqueous buffers of varying 2H2O content, we have examined the specific cro repressor-OR3 DNA complex in detail. The results are interpreted in the context of the popular model for cro repressor-OR3 complex derived from the three-dimensional structure of the cro repressor in the absence of DNA. The results presented here not originally predicted by the model are: (1) there is an asymmetry in the environment at the two ends of the operator, although the base-pairs involved and the cro repressor dimer are symmetric; (2) there appears to be distortion of the DNA helix at two distinct positions; (3) changes of the DNA environment in the middle of the helix suggest additional DNA distortion not near the contact areas proposed in the model. 相似文献
3.
The molten globule state of equine lysozyme is more stable than that of alpha-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105-110) in human alpha-lactalbumin with the helix D (residues 109-114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human alpha-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of alpha-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions. 相似文献
4.
Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor 总被引:18,自引:0,他引:18
It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein. 相似文献
5.
The technique of 1H NMR spectroscopy and absorption UV spectroscopy were used to study the ionization of the tyrosine phenol cycles and the effect of ionizable groups on the chemical shifts of signals from protons in the side chains of several amino acid residues. The microenvironment of these residues was established by analysing the titration curves. The mutual orientation of two functionally important adjacent alpha-helical protein regions was determined in solution. The signals from methionine residues belonging to different regions of the secondary structure were assigned in the NMR spectrum. The results indicate that the spatial structure of the repressor is similar in solution an in the crystal. They confirm the model proposed for the cro repressor interaction with DNA and based on the data of X-ray diffraction analysis. 相似文献
6.
S C Harrison J E Anderson G B Koudelka A Mondragon S Subbiah R P Wharton C Wolberger M Ptashne 《Biophysical chemistry》1988,29(1-2):31-37
The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA. 相似文献
7.
A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements. 相似文献
8.
The involvement of molten globule state as a distinct intermediate in the denaturation process in proteins is well documented. However, the structural characterization of such an intermediate is far from complete. We have, using fluorescence and fluorescence quenching, studied the molten globule state of bovine alpha-lactalbumin. Unlike the native state, where all the 4 tryptophans are buried in the protein, 2 tryptophans are exposed in the molten globule state. Using the hydrophobic photoactivable reagent [3H]diazofluorene, we observe an increased hydrophobic exposure in the molten globule state. These structural characteristics conform to the current views on the molten globule state, i.e. it has similar secondary structure but a poorly defined tertiary structure. Our fluorescence studies indicate the involvement of a premolten globule state in the native to molten globule state transition. This premolten globule state exists at pH 5.0 and has a very compact structure involving increased hydrophobic interactions in the protein interior. These results are also supported by circular dichroism studies. 相似文献
9.
The OR control system of bacteriophage lambda. A physical-chemical model for gene regulation 总被引:5,自引:0,他引:5
A quantitative model has been developed for processes in the bacteriophage lambda that control the switchover from lysogenic to lytic modes of growth. These processes include the interactions of cI repressor and cro proteins at the three DNA sites of the right operator, OR, the binding of RNA polymerase at promoters PR and PRM, the synthesis of cI repressor and cro proteins, and the degradative action of recA during induction of lysis. The model is comprised of two major physical-chemical components: a statistical thermodynamic theory for relative probabilities of the various molecular configurations of the control system; and a kinetic model for the coupling of these probabilities to functional events, including synthesis of regulatory proteins cI and cro. Using independently evaluated interaction constants and rate parameters, the model was found capable of predicting essential physiological characteristics of the system over an extended time. Sufficiency of the model to predict known physiological properties lends credence to the physical-chemical assumptions used in its construction. Several major physiological characteristics were found to arise as "system properties" through the non-linear, time-dependent, feedback-modulated combinations of molecular interactions prescribed by the model. These include: maintenance of the lysogenic state in the absence of recA-mediated cI repressor degradation; induction of lysis and the phenomenon of subinduction; and autogenous negative control of cro. We have used the model to determine the roles, within the composite system, of several key molecular processes previously characterized by studies in vitro. These include: co-operativity in cI repressor binding to DNA; interactions between repressors and RNA polymerase (positive control); and the monomer-dimer association of cI repressor molecules. A major role of cI repressor co-operativity is found to be that of guaranteeing stability of the lysogenic state against minor changes in cI repressor levels within the cell. The role of positive control seems to be that of providing for a peaked, rather than monotonic, dependence of PRM activity on cI repressor level, while permitting PR activity to be a step function. The model correlates an immense body of studies in vivo and in vitro, and it makes testable predictions about molecular phenomena as well as physiological characteristics of bacteriophage lambda. The approach developed in this study can be extended to include more features of the lambda system and to treat other systems of gene regulation. 相似文献
10.
Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding. 相似文献
11.
The interaction of the recognition helix of lac repressor with lac operator. 总被引:25,自引:12,他引:13
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N Lehming J Sartorius M Niemller G Genenger B v Wilcken-Bergmann B Müller-Hill 《The EMBO journal》1987,6(10):3145-3153
We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor. 相似文献
12.
Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme. 总被引:2,自引:2,他引:0
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S. Bhattacharjya P. Balaram 《Protein science : a publication of the Protein Society》1997,6(5):1065-1073
A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins. 相似文献
13.
The molten globule state of cytochrome c is the major intermediate of protein folding. Elucidation of the thermodynamic mechanism of conformational stability of the molten globule state would enhance our understanding of protein folding. The formation of the molten globule state of cytochrome c was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS, at low concentrations. The refolding states of the protein were monitored by spectroscopic techniques including circular dichroism (CD), visible absorbance and fluorescence. The effect of n-alkyl sulfates on the structure of acid-unfolded horse cytochrome c at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of the molten globule state. The addition of n-alkyl sulfates to the unfolded state of cytochrome c appears to support the stabilized form of the molten globule. The m-values of the refolded state of cytochrome c by SOS, SDeS, SDS, and STS showed substantial variation. The enhancement of m-values as the stability criterion of the molten globule state corresponded with increasing chain length of the cited n-alkyl sulfates. The compaction of the molten globule state induced by SDS, as a prototype for other n-alkyl sulfates, relative to the unfolded state of cytochrome c was confirmed by Stokes radius and thermal transition point (T(m)) measured by microviscometry and differential scanning calorimetry (DSC), respectively. Thus, hydrophobic interactions play an important role in stabilizing the molten globule state. 相似文献
14.
A model of dynamic side-chain--side-chain interactions in the alpha-lactalbumin molten globule
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Proteins in the molten globule state contain high levels of secondary structure, as well as a rudimentary, nativelike tertiary topology. Thus, the structural similarity between the molten globule and native proteins may have a significant bearing in understanding the protein-folding problem. To explore the nature of side-chain--side-chain interactions in the alpha-lactalbumin (alpha-LA) molten globule, we determined the effective concentration for formation of the 28--111 disulfide bond in 14 double-mutant proteins, each containing two hydrophobic core residues replaced by alanine. We compared our results with those of single-alanine substitutions using the framework of double-mutant cycle analysis and found that, in the majority of cases, the effects of two alanine substitutions are additive. Based on these results, we propose a model of side-chain-side-chain interactions in the alpha-LA molten globule, which takes into consideration the dynamic nature of this partially folded species. 相似文献
15.
The molten globule state of alpha-lactalbumin (alpha-LA) has been considered a prototype of partially folded proteins. Despite the importance of molten globules in understanding the mechanisms of protein folding and its relevance to some biological phenomena, site-specific information on the structure and dynamics of a molten globule is limited, largely because of the high conformational flexibility and heterogeneity. Here, we use selective isotope labeling and (19)F NMR to investigate the solvent accessibility and side-chain dynamics of aromatic residues in the molten globule of alpha-LA. Comparison of these properties with those of the native and unfolded protein indicates that the alpha-LA molten globule is highly heterogeneous; each residue has its unique solvent accessibility and motional environment. Many aromatic residues normally buried in the interior of native alpha-LA remain significantly buried in the molten globule and the side-chain dynamics of these residues are highly restricted. Our results suggest that hydrophobic and van der Waals interactions mediated by the inaccessible surface area could be sufficient to account for all the stability of the alpha-LA molten globule, which is approximately 50% of the value for the native protein. 相似文献
16.
17.
G V Gursky A N Surovaya A V Kurochkin B K Chernov S K Volkov M P Kirpichnikov 《Journal of biomolecular structure & dynamics》1992,10(1):15-33
In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F. 相似文献
18.
The nucleotide sequence of the operators of ultravirulent mutants of lambda, able to grow on host cells with elevated repressor levels, was determined. It appears that ultravirulence in lambda requires multiple mutational events at the operator sequences. OL1, OL2, and OL3 operator sites are the target of mutational changes in ultravirulent phages indicating that these sites participate in vivo in repression of the PL promoter. No changes were found in the OR3 sequence, in contrast there is a mutation in OR2 and two mutations in OR1, in both lambda 668 and lambda 2668 phages. This mutated operator structure accounts for the constitutive expression of their PR promoter either in cells overproducing the lambda repressor or in cells overproducing the cro gene product. A model of the structure of the lambda operator site is proposed. The nucleotide sequence in each site can be divided into two functionally different subsets, one of which is recognized by the repressor while the other stabilizes the repressor-operator interaction. 相似文献
19.
The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242. 相似文献
20.
P Hammarstr?m M Persson P O Freskgard L G Martensson D Andersson B H Jonsson U Carlsson 《The Journal of biological chemistry》1999,274(46):32897-32903
Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large beta-sheet of the protein, within a limited region between the central beta-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific beta-sheet containing regions in aggregate formation. 相似文献