A helical capping motif in ShK toxin and its role in helix stabilization

Lanigan, Mark D.; Tudor, Jane E.; Pennington, Michael W.; Norton, Raymond S.

doi:10.1002/1097-0282(20010405)58:4<422::aid-bip1018>3.0.co;2-t

Cited by 11 publications

(11 citation statements)

References 41 publications

(53 reference statements)

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“…They are sometimes involved in the helix capping process via hydrogen bonding between R-helix residues and flanking residues (24)(25)(26)(27)(28). Some proteins are stabilized by capping motifs, and the fraying of R-helices is prevented (24). While there is no chance to form a C-terminal cap (C-cap) at the end of the E-helix at pH 8.0, the protonation of the imidazole side chain of His82 in the F at pH 4.2 can provide a proton donor, and thereby the His82 can form a hydrogen bond with the terminal backbone of helix E. Therefore, enzymatic accessibility to Glu83 becomes restricted by the local rigidity of the newly formed C-cap in acidic environment.…”

Section: Resultsmentioning

confidence: 99%

Structural Characterization of the Molten Globule State of Apomyoglobin by Limited Proteolysis and HPLC-Mass Spectrometry

Kim

Park

et al. 2005

Biochemistry

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A method to characterize the structural conformation of an acidic molten globule apomyoglobin (apoMb) at pH 4.2 was developed using limited proteolysis and HPLC-mass spectrometry (HPLC-MS). Endoproteinase Glu-C, which has a double maximum activity at pH 4.0 and pH 7.8 toward glutamic acid (Glu), was used as a proteolytic enzyme. Using this method enabled us to compare the proteolytic cleavages of native apoMb (at pH 8.0) and molten globule (at pH 4.2) directly. Only the first cleavage event in each molecule was considered as reflecting original structural information since the original structure of the protein can be altered after the fist cleavage. Structural changes of apoMb in various pH conditions were studied here to elucidate the local helicity of molten globule apoMb. Among 13 Glu sites, only Glu83 and Glu85 in the F-helix were cleaved at pH 8.0, which confirms that only helix F is frayed upon removal of heme group. At acidic molten globule state, rapid cleavages at Glu38, Glu52, Glu54, Glu85, and Glu148 were detected, while the remaining eight sites were protected. Glu6 and Glu18 in the A-helix, and Glu105 in the G-helix were protected due to the helicity of the secondary structures. The cleavage at Glu38 and the protection at Glu41 in the C-helix indicate that the first half of the C-helix is frayed and the second half of the C-helix is structured. Cleavage at both Glu52 and Glu54 in the D-helix proves that the D-helix is disordered. The N-terminal end of the E-helix at Glu59 was protected, and the beginning of the F-helix was protected by aid of the pH-induced C-cap of the E-helix. The cleavage at Glu148 in H suggests that the C-terminal end of the H-helix is disordered. The A-helix and the first half of the B-helix were highly stable.

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Section: Resultsmentioning

confidence: 99%

Structural Characterization of the Molten Globule State of Apomyoglobin by Limited Proteolysis and HPLC-Mass Spectrometry

Kim

Park

et al. 2005

Biochemistry

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“…Our secondary structure assignment is consistent with known capping motifs. T13 and Q16 provides an N-terminal helix capping motif in ShK toxin, which plays a role in helix stabilization [47]. CR I -II (T17 and Q20) and Calb I -II (T12 and Q16) contain analogous residues at the N-terminus of helix A.…”

Section: Definition Of Cr I -Ii Structurementioning

confidence: 99%

Structural and biochemical characterization of neuronal calretinin domain I–II (residues 1–100)

Palczewska

Groves

Ambrus

et al. 2001

European Journal of Biochemistry

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This study characterizes the calcium‐bound CR I–II domain (residues 1–100) of rat calretinin (CR). CR, with six EF‐hand motifs, is believed to function as a neuronal intracellular calcium‐buffer and/or calcium‐sensor. The secondary structure of CR I–II, defined by standard NMR methods on 13C,15N‐labeled protein, contains four helices and two short interacting segments of extended structure between the calcium‐binding loops. The linker between the two helix–loop–helix, EF‐hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I–II) confirms that the linker of CR I–II is solvent‐exposed and that other potential sites are protected by regular secondary structure. 45Ca‐overlay of glutathione S‐transferase (GST)–CR(1–60) and GST–CR(61–100) fusion proteins confirm that both EF‐hands of CR I–II have intrinsic calcium‐binding properties. The primary sequence and NMR chemical shifts, including calcium‐sensitive glycine residues, also suggest that both EF‐hand loops of CR I–II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross‐linking and NMR translation diffusion measurements indicate that CR I–II exists as a monomer. Calb I–II (the homologous domain of calbindin D28k) has the same EF‐hand secondary structures as CR I–II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem.262, 933–938]. In contrast, Calb I–II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I–II and Calb I–II probably have distinct tertiary structure features that suggest different cellular functions for the full‐length proteins.

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“…The Ncap residue is serine in R2. c The NOE connectivities were reported for the N-capping box by Lanigan et al(50). The plus signs indicate the NOE connectivities observed in a 200 ms mixing time NOESY spectrum.…”

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confidence: 95%

Structural Approach to a Novel Tandem Repeat DNA-Binding Domain, STPR, by CD and NMR^,

et al. 2007

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Fibroin-modulator-binding protein 1 (FMBP-1) is a factor that binds the transcriptional activation elements of the fibroin gene. It has a novel structure, consisting of four tandem repeats (R1-R4) of 23 amino acids each in the C-terminal half. This region is referred to as the STPR (score and three amino acid peptide repeat) domain and acts as a DNA-binding domain in FMBP-1. Interestingly, the homology among the four repeats is remarkably high. Here, we have determined the three-dimensional structures of the four repeats by NMR. All four repeat units have basically the same structure: a short alpha-helix in the N-terminal half maintained by a salt bridge and an N-capping box. CD studies showed that the full-length STPR domain was 31% helical in solution. This is explained by the connections among the four short helices that were determined separately by NMR. From the thermal-denaturation study, it can be deduced that these four helices in the full-length STPR domain moved flexibly with no interaction among them. However, the specific DNA caused a distinct increase, of up to 76%, in the alpha-helical content of the full-length STPR domain. This finding suggests that the binding of the full-length STPR domain to specific DNA causes an induced-fit conformational change that increases alpha-helicity; the poorly structured regions of the protein may form a regular secondary structure. Furthermore, the mutation analysis showed that the four repeats of the STPR domain raise the possibility of interaction with DNA in different ways.

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A helical capping motif in ShK toxin and its role in helix stabilization

Cited by 11 publications

References 41 publications

Structural Characterization of the Molten Globule State of Apomyoglobin by Limited Proteolysis and HPLC-Mass Spectrometry

Structural Characterization of the Molten Globule State of Apomyoglobin by Limited Proteolysis and HPLC-Mass Spectrometry

Structural and biochemical characterization of neuronal calretinin domain I–II (residues 1–100)

Structural Approach to a Novel Tandem Repeat DNA-Binding Domain, STPR, by CD and NMR^,

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A helical capping motif in ShK toxin and its role in helix stabilization

Cited by 11 publications

References 41 publications

Structural Characterization of the Molten Globule State of Apomyoglobin by Limited Proteolysis and HPLC-Mass Spectrometry

Structural Characterization of the Molten Globule State of Apomyoglobin by Limited Proteolysis and HPLC-Mass Spectrometry

Structural and biochemical characterization of neuronal calretinin domain I–II (residues 1–100)

Structural Approach to a Novel Tandem Repeat DNA-Binding Domain, STPR, by CD and NMR,

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Structural Approach to a Novel Tandem Repeat DNA-Binding Domain, STPR, by CD and NMR^,