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Biomedical subjects

M G Hinds

Publications and source records attributed to M G Hinds.

At least 19 recordsLinked to original sources
PubMed2006

Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets.

All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance.

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.

Amino Acid Sequence↗
PubMed2000

Backbone dynamics measurements on leukemia inhibitory factor, a rigid four-helical bundle cytokine.

The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine-human chimera, MH35-LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D(parallel)/D(perpendicular) of 1.31 and an effective correlation time (4D(perpendicular) + 2D(parallel))(-1) of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 degrees C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.

Amides↗
PubMed1999

1H NMR study of robustoxin, the lethal neurotoxin from the funnel web spider Atrax robustus.

Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.

Amino Acid Sequence↗
PubMed1999

Solution structure of a baculoviral inhibitor of apoptosis (IAP) repeat.

Members of the inhibitor of apoptosis (IAP) family of proteins are able to inhibit cell death following viral infection, during development or in cell lines in vitro. All IAP proteins bear one or more baculoviral IAP repeats (BIRs). Here we describe the solution structure of the third BIR domain from the mammalian IAP homolog B (MIHB/c-IAP-1). The BIR domain has a novel fold that is stabilized by zinc tetrahedrally coordinated by one histidine and three cysteine residues. The structure consists of a series of short alpha-helices and turns with the zinc packed in an unusually hydrophobic environment created by residues that are highly conserved among all BIRs.

Amino Acid Sequence↗
PubMed1999

Solution structure and mutagenesis of the caspase recruitment domain (CARD) from Apaf-1.

Activation of procaspase-9, a key component of the apoptosis mechanism, requires the interaction of its caspase recruitment domain (CARD) with the CARD in the adaptor protein Apaf-1. Using nuclear magnetic resonance spectroscopy and mutagenesis we have determined the structure of the CARD from Apaf-1 and the residues important for binding the CARD in procaspase-9. Apaf-1's CARD contains seven short alpha-helices with the core six helices arranged in an antiparallel manner. Residues in helix 2 have a central role in mediating interaction with procaspase-9 CARD. This interaction surface is distinct from that proposed based on the structure of the CARD from RAIDD, but is coincident with that of the structurally similar FADD death effector domain and the Apaf-1 CARD interface identified by crystallographic studies.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Solution structure of leukemia inhibitory factor.

The solution structure of a murine-human chimera of leukemia inhibitory factor (LIF), a 180-residue cytokine with a molecular mass of 20 kDa, has been determined using multidimensional heteronuclear NMR techniques. The protein contains four alpha-helices, the relative orientations of which are well defined on the basis of long-range interhelical nuclear Overhauser effects. The helices are arranged in an up-up-down-down orientation, as found in other four-helix bundle cytokines, and the overall topology of the chimera is similar to that of the crystal structure of murine LIF (Robinson, R. C., Grey, L. M., Staunton, D., Vankelecom, H. Vernallis, A. B., Moreau, J. F., Stuart, D. I., Heath, J. K., and Jones, E. Y. (1994) Cell 77, 1101-1116). Differences between the structures are evident in the N-terminal region, where the peptide bond preceding Pro17 has a trans-conformation in solution but a cis-conformation in the crystal, and in the small antiparallel beta-sheet encompassing residues in the N terminus and the CD loop in the crystal structure, which is not apparent in solution. There are also minor differences in the extent of the helices. Other than at the N terminus, the main difference between the two structures occurs at the C-terminal end of the CD loop. As this loop is close to a receptor-binding site on LIF that makes a major contribution to high affinity binding to the LIF receptor alpha-chain, these differences between the solution and crystal structures should be taken into account in structural models of LIF receptor interactions.

Amino Acid Sequence↗
PubMed1997

Solution structure of a polypeptide from the N terminus of the HIV protein Nef.

Nef is a 27 kDa myristylated phosphoprotein expressed early in infection by HIV. The N terminus of Nef is thought to play a vital role in the functions of this protein through its interactions with membrane structures. The solution structure of a 25-residue polypeptide corresponding to the N terminus of Nef (Nef1-25) has been investigated by 1H NMR spectroscopy. In aqueous solution at pH 4.8 and 281 K, this peptide underwent conformational averaging, with Pro13 existing in cis and trans conformations in nearly equal proportions. In methanol solution, however, the peptide adopted a well-defined alpha-helical structure from residues 6 to 22, with the N- and C-terminal regions having a less ordered structure. On the basis of a comparison of chemical shifts and NOEs, it appeared that this helical structure was maintained in aqueous trifluoroethanol (50% v/v) and to a lesser extent in a solution of SDS micelles. When the N-acetyl group was replaced by either an N-myristyl or a free ammonium group, there was little effect on the three-dimensional structure of the peptide in methanol; deamidation of the C terminus also had no effect on the structure in methanol. In water, the myristylated peptide aggregated. The similarity between the sequences of Nef1-25 and melittin is reflected in the similar structures of the two molecules, although the N-terminal helix of melittin is more defined. This similarity in structure raises the possibility that Nef1-25 not only interacts with membranes but also may be capable of disrupting them and causing cell lysis. This type of interaction could contribute at least in part to the killing of bystander cells in lymphoid tissues during HIV infection.

Amino Acid Sequence↗
PubMed1997

NMR spectroscopy of peptides and proteins. Practical considerations.

High resolution nuclear magnetic resonance (NMR) spectroscopy is the only method available for determining the three-dimensional structures of peptides and proteins in solution at atomic resolution. This article deals with a range of practical considerations associated with such studies, including sample preparation, instrumental setup, one- and two-dimensional NMR methods, interpretation of spectral data, and structure calculations.

Animals↗
PubMed1997

Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution.

The chemical shift assignments and secondary structure of a murine-human chimera, MH35, of leukaemia inhibitory factor (LIF), a 180-residue protein of molecular mass 20 kDa, have been determined from multidimensional heteronuclear NMR spectra acquired on a uniformly 13C, 15N-labelled sample. Secondary structure elements were defined on the basis of chemical shifts, NH-C alpha H coupling constants; medium-range NOEs and the location of slowly exchanging amide protons. The protein contains four alpha-helices, the relative orientations of which were determined on the basis of long-range, interhelical NOEs. The four helices are arranged in an up-up-down-down orientation, as found in other four-helical bundle cytokines. The overall topology of MH35-LIF is similar to that of the X-ray crystallographic structure for murine LIF [Robinson et al. (1994) Cell, 77, 1101-1116]. Differences between the X-ray structure and the solution structure are evident in the N-terminal tail, where the solution structure has a trans-Pro17 compared with the cis-Pro17 found in the crystal structure and the small antiparallel beta-sheet encompassing residues in the N-terminus and CD loop in the crystal structure is less stable.

Amino Acid Sequence↗
PubMed1995

Solution properties of Escherichia coli-expressed VH domain of anti-neuraminidase antibody NC41.

The VH domain of anti-influenza neuraminidase antibody NC41, with and without a C-terminal hydrophilic marker peptide (FLAG), has been expressed in high yield (15-27 mg/L) in Escherichia coli. Both forms were secreted into the periplasm where they formed insoluble aggregates which were solubilized quantitatively with 2 M guanidine hydrochloride and purified to homogeneity by ion-exchange chromatography. The VH-FLAG was composed of three isoforms (pI values of approximately 4.6, 4.9, and 5.3) and the VH molecule was composed of two isoforms with pI values of 5.1 and 6.7; the difference between the VH isoforms was shown to be due to cyclization of the N-terminal glutamine residue in the pI 5.1 isoform. At 20 degrees C and concentrations of 5-10 mg/ml the VH domain dimerized in solution and then partly precipitated, resulting in the broadening of resonances in its 1H NMR spectrum. Reagents such as CHAPS, n-ocytylglucoside, and ethylene glycol, which presumably mask the exposed hydrophobic interface of the VH molecule, prevented dimerization of the VH and permitted good-quality NMR spectra on isotope-labeled protein to be obtained.

Amino Acid Sequence↗
PubMed1993

Conformational differences between complexes of elongation factor Tu studied 19F-NMR spectroscopy.

An analogue of elongation factor Tu (EF-Tu) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorotyrosine. The 19F-NMR spectra of the binary complexes of this protein with GDP, GTP and elongation factor Ts (EF-Ts) and the ternary complexes EF-Tu.GDP.aurodox and EF-Tu.GDP.EF-Ts were measured. EF-Tu contains ten tyrosine residues and all of the complexes studied gave complex 19F spectra with overlapping resonances. EF-Tu.GDP gave a spectrum in which two signals were markedly different from those shown by the other complexes, the two resonances being shifted downfield by at least 3.4 ppm and 0.9 ppm relative to their shifts in the other complexes. Such large downfield shifts can be explained by second-order electric field shielding effects resulting from these two tyrosine residues being in a sterically constrained environment in EF-Tu.GDP and with the steric restraints being released in all of the other complexes. The X-ray diffraction structure of EF-Tu.GDP shows that Tyr87 in the N-terminal domain (domain I) and Tyr309 in the C-terminal domain (domain III) are both buried within the protein and are close to each other: these residues are in regions of EF-Tu previously implicated in the structural changes between EF-Tu.GDP and EF-Tu.GTP by other workers. If these tyrosine residues correspond to the two downfield resonances of the spectra of EF-Tu.GDP, the results from the 19F-NMR would be consistent with these earlier indications that domain I interacts closely with domain III in EF-Tu.GDP and that the amino acids between Gly83 and Gly100 are an important part of this interaction. For all the other complexes studied, these tyrosines are in a less sterically crowded environment consistent with a weaker interaction between the two domains. The 19F-NMR spectrum of the trypsin-cleaved product of EF-Tu.GDP, from which the X-ray diffraction structural data have been obtained, shows no significant differences from the native protein so that trypsin cleavage causes no large changes in the protein's structure.

Aurodox↗
PubMed1993

Sequential 1H-NMR assignments of neurotoxin III from the sea anemone Heteractis macrodactylus and structural comparison with related toxins.

The complete sequence-specific assignment of resonances in the 1H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemone Heteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III from Heteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The C alpha H resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected C beta H resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7-16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I from Stichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.

Amino Acid Sequence↗
PubMed1992

19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli.

A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.

Bacterial Proteins↗