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1.
Protein folding: evaluation of some simple rules for the assembly of helices into tertiary structures with myoglobin as an example 总被引:11,自引:0,他引:11
A computer program designed to fold a peptide chain consisting solely of helical segments and connecting links of known length is described and evaluated. This study is a detailed extension of certain aspects of the earlier work of Ptitsyn &; Rashin (1975). Possible interaction sites on the helices are sequence dependent and are calculated as described by Richmond &; Richards (1978) using probable changes in solvent contact area as a guide. The helices are then paired according to the list of potential sites, with each helix being paired at least once. The lists of pairings are then examined geometrically, each site having a defined dihedral helix axis angle, a specified inter-helix axis distance, and defined rotations, when required, about each helix axis. Two simplified filters are used: (1) lengths of connecting links must be equal to or greater than the end-to-end distances of the helices; and (2) non-paired helices must not collide. With myoglobin as a test example and only six of the eight helices being considered, a conformation space consisting of more than 3 × 108 structures was surveyed. The two filters reduced the acceptable structure list to 121. Slight readjustment of the parameters in the filters would have reduced this to 20 structures. Of these 20, one closely resembles the actual distribution of helices in myoglobin. The possible utility and pitfalls of this approach as part of an overall protein folding program are discussed. 相似文献
2.
Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2′-endo region and the phosphodiester conformations are (trans, gauche?), and the RNA helices have sugars in the C3′-endo region and the phosphodiester conformations are (gauche?, gauche?). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix. © 1994 John Wiley & Sons, Inc. 相似文献
3.
Manju Bansal Sandeep Kumart R. Velavan 《Journal of biomolecular structure & dynamics》2013,31(5):811-819
Abstract A detailed analysis of structural and position dependent characteristic features of helices will give a better understanding of the secondary structure formation in globular proteins. Here we describe an algorithm that quantifies the geometry of helices in proteins on the basis of their Cα atoms alone. The Fortran program HELANAL can extract the helices from the PDB files and then characterises the overall geometry of each helix as being linear, curved or kinked, in terms of its local structural features, viz. local helical twist and rise, virtual torsion angle, local helix origins and bending angles between successive local helix axes. Even helices with large radius of curvature are unambiguously identified as being linear or curved. The program can also be used to differentiate a kinked helix and other motifs, such as helix-loop-helix or a helix-turn-helix (with a single residue linker) with the help of local bending angles. In addition to these, the program can also be used to characterise the helix start and end as well as other types of secondary structures. 相似文献
4.
Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study 总被引:6,自引:0,他引:6
Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c2 were studied by1H–15N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe82 to Asp87 are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His17, Leu37, Arg43, Ala45, Gly46, Ile57, Val58, Leu60, Gly61 and Leu100 suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR
nuclear magnetic resonance
- HMQC
homonuclear multiple quantum correlation
- NOESY
nuclear Overhauser effect spectroscopy 相似文献
5.
Yuichiro Fujiwara Tatsuki Kurokawa Yasushi Okamura 《The Journal of general physiology》2014,143(3):377-386
The voltage-gated H+ channel (Hv) is a H+-permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1–S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively, which is mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. However, the structural basis of the interdomain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, suggesting that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. The interaction interface between the two S4 and the assembly interface of the coiled-coil domain were aligned in the same direction based on the phase angle calculation along α helices. Collectively, we propose that continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer. 相似文献
6.
Human upstream binding factor (hUBF) HMG Box‐5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA‐binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α‐helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three‐state thermal unfolding equilibrium of hUBF HMG Box‐5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box‐5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box‐5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box‐5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential 1HNi‐1HNi+1 NOE analyses indicate that helices 1 and 2 are native‐like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix‐turn‐helix folding model of Box‐5, where helices 1 and 2 potentially form the helix 1‐turn‐helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley‐Liss, Inc. 相似文献
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8.
Zhou Y Madej MG Guan L Nie Y Kaback HR 《The Journal of biological chemistry》2011,286(35):30415-30422
Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg144 and Trp151, two residues that play direct roles in sugar recognition and binding, as well as Cys154, which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro28 → Cys/Cys154, Pro28 → Cys/Val158 → Cys, and Phe29 → Cys/Val158 → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)2-1), 3–4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)2-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding. 相似文献
9.
E. Peggion S. Mammi E. Schievano R. P. Revoltella C. Galoppini P. Rovero 《Biopolymers》1999,50(5):545-554
An analogue of the human granulocyte–macrophage colony‐stimulating factor (hGM‐CSF), hGM‐CSF(13–27)–Gly–(75–87) was synthesized by solid phase methodology. This analogue was designed to comprise helices A and C of the native growth factor, linked by a glycine bridge. Helices A and C form half of a four‐helix bundle motif in the crystal structure of the native factor and are involved in the interaction with α‐ and β‐chains of the heterodimeric receptor. A conformational analysis of the synthetic analogue by CD, two‐dimensional nmr spectroscopy, and molecular dynamics calculations is reported. The analogue is in a random structure in water and assumes a partially α‐helical conformation in a 1 : 1 trifluoroethanol/water mixture. The helix content in this medium is ∼ 70%. By 2D‐nmr spectroscopy, two helical segments were identified in the sequences corresponding to helices A and C. In addition to medium‐ and short‐range NOESY connectivities, a long‐range cross peak was found between the Cβ proton of Val16 and NH proton of His87 (using the numbering of the native protein). Experimentally derived interproton distances were used as restraints in molecular dynamics calculations, utilizing the x‐ray coordinates as the initial structure. The final structure is characterized by two helical segments in close spatial proximity, connected by a loop region. This structure is similar to that of the corresponding domain in the x‐ray structure of the native growth factor in which helices A and C are oriented in an antiparallel fashion. The N‐terminal residues Gly–Pro of helix C are involved in an irregular turn connecting the two helical segments. As a consequence, helix C is appreciably shifted and slightly rotated with respect to helix A compared to the x‐ray structure of the native growth factor. These small differences in the topology of the two helices could explain the lower biological activity of this analogue with respect to that of the native growth factor. © 1999 John Wiley & Sons, Inc. Biopoly 50: 545–554, 1999 相似文献
10.
小型核酶的结构和催化机理 总被引:5,自引:1,他引:4
自然界存在的小型核酶主要有锤头型核酶、发夹型核酶、肝炎δ病毒(HDV)核酶和VS核酶.锤头型核酶由3个短螺旋和1个广义保守的连接序列组成;发夹型核酶的催化中心由两个肩并肩挨着的区域构成;HDV核酶折叠成包含五个螺旋臂(P1~P4)的双结结构;VS核酶由五个螺旋结构组成,这些螺旋结构通过两个连接域连接起来.小型核酶的催化机理与其分子结构密切相关.金属离子或特定碱基都可作为催化反应的关键成分.锤头型核酶的催化必须有金属离子(尤其是二价金属离子)参与,而发夹型核酶则完全不需要金属离子.基因组HDV核酶进行催化时要有金属离子和特定碱基互相配合. 相似文献
11.
A computer method for finding common base paired helices in aligned sequences: application to the analysis of random sequences.
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We describe a new computer program that identifies conserved secondary structures in aligned nucleotide sequences of related single-stranded RNAs. The program employs a series of hash tables to identify and sort common base paired helices that are located in identical positions in more than one sequence. The program gives information on the total number of base paired helices that are conserved between related sequences and provides detailed information about common helices that have a minimum of one or more compensating base changes. The program is useful in the analysis of large biological sequences. We have used it to examine the number and type of complementary segments (potential base paired helices) that can be found in common among related random sequences similar in base composition to 16S rRNA from Escherichia coli. Two types of random sequences were analyzed. One set consisted of sequences that were independent but they had the same mononucleotide composition as the 16S rRNA. The second set contained sequences that were 80% similar to one another. Different results were obtained in the analysis of these two types of random sequences. When 5 sequences that were 80% similar to one another were analyzed, significant numbers of potential helices with two or more independent base changes were observed. When 5 independent sequences were analyzed, no potential helices were found in common. The results of the analyses with random sequences were compared with the number and type of helices found in the phylogenetic model of the secondary structure of 16S ribosomal RNA. Many more helices are conserved among the ribosomal sequences than are found in common among similar random sequences. In addition, conserved helices in the 16S rRNAs are, on the average, longer than the complementary segments that are found in comparable random sequences. The significance of these results and their application in the analysis of long non-ribosomal nucleotide sequences is discussed. 相似文献
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The Na+-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na+-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state. 相似文献
14.
The Na/Ca-K exchanger (NCKX) is a polytopic membrane protein that plays a critical role in Ca(2+) homeostasis in retinal rod and cone photoreceptors. The NCKX1 isoform is found in rods, while the NCKX2 isoform is found in cones, in retinal ganglion cells, and in various parts of the brain. The topology of the Na/Ca-K exchanger is thought to consist of two large hydrophilic loops and two sets of transmembrane spanning segments (TMs). The first large hydrophilic loop is located extracellularly at the N-terminus; the other is cytoplasmic and separates the two sets of TMs. The TMs consist of either five and five membrane spanning helices or five and six membrane spanning helices, depending upon the predictive algorithm used. Little specific information is yet available on the orientation of the various membrane spanning helices and the localization of the short loops connecting these helices. In this study, we have determined which of the connecting loops are exposed to the extracellular milieu using two different methods: accessibility of substituted cysteine residues and insertion of N-glycosylation sites. The two methods resulted in a consistent NCKX topology in which the two sets of TMs each contain five membrane spanning helices. Our new model places what was previously membrane spanning helix six in the cytoplasm, which places the C-terminus on the extracellular surface. Surprisingly, this NCKX topology model is different from the current NCX topology model with respect to the C-terminal three membrane helices. 相似文献
15.
The solution conformation of the antibiotic peptide alamethicin was investigated using multi-nuclear spectroscopy and the distance geometry/simulated annealing algorithms from the program DSPACE. 1H-, 13C-, and 15N-nmr chemical shifts and homonuclear 1H coupling constants suggest that the molecule is flexible in the vicinity of Gly-11 and Leu-12. The temperature dependence of the amide proton chemical shifts indicates that there is flexibility in the middle of the 20 residue peptide and provides evidence that, at the very N-terminus, the molecule adopts a 310-helical conformation. The large differences in the 13C chemical shifts of the pro-R and pro-S methyls of the α-aminoisobutyric acid residues were used to constrain those residues to the right-handed helical conformation in the distance geometry/simulated annealing algorithms. A family of 24 structures was generated but did not converge to a common conformation when superimposed over the entire polypeptide sequence. The molecules did converge to a helical conformation over residues 1–10 and residues 13–18. The lack of convergence when the entire lengths of the molecules are superimposed is explained by the flexibility of the peptide near Gly-11/Leu-12. The results suggest that the protein consists of two helices connected by a flexible “hinge.” The flexibility of the molecule is discussed with respect to the macrodipole model of voltage gating. © 1995 John Wiley & Sons, Inc. 相似文献
16.
Jesus Torres-Bacete Prem Kumar Sinha Norma Castro-Guerrero Akemi Matsuno-Yagi Takao Yagi 《The Journal of biological chemistry》2009,284(48):33062-33069
The bacterial H+-pumping NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzymatic complex. Escherichia coli NDH-1 is composed of 13 subunits (NuoA–N). NuoM (ND4) subunit is one of the hydrophobic subunits that constitute the membrane arm of NDH-1 and was predicted to bear 14 helices. We attempted to clarify the membrane topology of NuoM by the introduction of histidine tags into different positions by chromosomal site-directed mutagenesis. From the data, we propose a topology model containing 12 helices (helices I–IX and XII–XIV) located in transmembrane position and two (helices X and XI) present in the cytoplasm. We reported previously that residue Glu144 of NuoM was located in the membrane (helix V) and was essential for the energy-coupling activities of NDH-1 (Torres-Bacete, J., Nakamaru-Ogiso, E., Matsuno-Yagi, A., and Yagi, T. (2007) J. Biol. Chem. 282, 36914–36922). Using mutant E144A, we studied the effect of shifting the glutamate residue to all sites within helix V and three sites each in helix IV and VI on the function of NDH-1. Twenty double site-directed mutants including the mutation E144A were constructed and characterized. None of the mutants showed alteration in the detectable levels of expressed NuoM or on the NDH-1 assembly. In addition, most of the double mutants did not restore the energy transducing NDH-1 activities. Only two mutants E144A/F140E and E144A/L147E, one helix turn downstream and upstream restored the energy transducing activities of NDH-1. Based on these results, a role of Glu144 for proton translocation has been discussed. 相似文献
17.
Solution NMR techniques are used to determine the structure and the topology of micelle integration of a large fragment of the Y4 receptor, a human G-protein-coupled receptor, that contains the entire N-terminal domain plus the first two transmembrane (TM) segments. The structure calculations reveal that the putative TM helices are indeed helical to a large extent, but that interruptions of secondary structure occur close to internal polar or charged residues. This view is supported by 15N relaxation data, amide-water exchange rates, and attenuations from micelle-integrating spin labels. No contacts between different helices are observed. This is in contrast to a similar TM1-TM2 fragment from the yeast Ste2p receptor for which locations of the secondary and the tertiary structure agreed well with the predictions from a homology model. The difference in structure is discussed in terms of principal biophysical properties of residues within central regions of the putative TM helices. Overall, using the biophysical scale of Wimley and White the TM regions of Ste2p display much more favorable free energies for membrane integration. Accordingly, the full secondary structure and the tertiary structure in TM1-TM2 of the Y4 receptor is likely to be formed only when tertiary contacts with other TM segments are created during folding of the receptor. 相似文献
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Voltage-gated K+ channels share a common voltage sensor domain (VSD) consisting of four transmembrane helices, including a highly mobile S4 helix that contains the major gating charges. Activation of ether-a-go-go (EAG) family K+ channels is sensitive to external divalent cations. We show here that divalent cations slow the activation rate of two EAG family channels (Kv12.1 and Kv10.2) by forming a bridge between a residue in the S4 helix and acidic residues in S2. Histidine 328 in the S4 of Kv12.1 favors binding of Zn2+ and Cd2+, whereas the homologous residue Serine 321 in Kv10.2 contributes to effects of Mg2+ and Ni2+. This novel finding provides structural constraints for the position of transmembrane VSD helices in closed, ion-bound EAG family channels. Homology models of Kv12.1 and Kv10.2 VSD structures based on a closed-state model of the Shaker family K+ channel Kv1.2 match these constraints. Our results suggest close conformational conservation between closed EAG and Shaker family channels, despite large differences in voltage sensitivity, activation rates, and activation thresholds. 相似文献
20.
G. Brandolin A. Le Saux V. Trezeguet G. J. M. Lauquin P. V. Vignais 《Journal of bioenergetics and biomembranes》1993,25(5):459-472
In the process of oxidative phosphorylation, the exchange of cytosolic ADP3– against mitochondrial ATP4– across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport. 相似文献