Biomedical subjects
Publications and source records attributed to B D Sykes.
Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice.
A computer program has been developed to accurately and automatically predict the 1H and 13C chemical shifts of unassigned proteins on the basis of sequence homology. The program (called SHIFTY) uses standard sequence alignment techniques to compare the sequence of an unassigned protein against the BioMagResBank--a public database containing sequences and NMR chemical shifts of nearly 200 assigned proteins [Seavey et al. (1991) J Biomol. NMR, 1, 217-236]. From this initial sequence alignment, the program uses a simple set of rules to directly assign or transfer a complete set of 1H or 13C chemical shifts (from the previously assigned homologues) to the unassigned protein. This 'homologous assignment' protocol takes advantage of the simple fact that homologous proteins tend to share both structural similarity and chemical shift similarity. SHIFTY has been extensively tested on more than 25 medium-sized proteins. Under favorable circumstances, this program can predict the 1H or 13C chemical shifts of proteins with an accuracy far exceeding any other method published to date. With the exponential growth in the number of assigned proteins appearing in the literature (now at a rate of more than 150 per year), we believe that SHIFTY may have widespread utility in assigning individual members in families of related proteins, an endeavor that accounts for a growing portion of the protein NMR work being done today.
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TGF-alpha, a 50 amino acid growth factor containing 3 disulfide bonds, was fused to the N-terminal domain of the pIII protein of fusN, a derivative of phagemid fd-tet, to form a TGF-alpha phage. The fusion phage showed binding activity to epidermal growth factor receptor (EGFR). A library of approximately 4 x 10(7) variants of TGF-alpha was generated with substitutions of total of 10 amino acids located in the C-loop region. This C-loop subdomain of TGF-alpha consists of a small antiparallel double hairpin structure involving interactions between intra-polypeptide segments. Mutants isolated from the phage library with greatly increased binding affinity were selected through panning with A431 cells (a cell line expressing an elevated number of EGFRs). Following two rounds of stringent selection, variant phages with higher binding affinity than wild type TGF-alpha were identified and the phage DNAs were sequenced for the alignment analysis. Absolute selection at position 42 as Arg, preferential selection at position 38 and 45 as Tyr or Phe with aromatic side chain and selection at position 41 with acidic residues, were obtained. Although an amino acid residue with smaller side chain at position 35 and one with larger side chain at position 36 were preferred, the steric hindering of the structure in side chains was minimized between these adjacent amino acids.
The nucleotide sequence encoding the N-terminal domain (residues 1-183) of human apolipoprotein E3 (apoE3) was cloned into the pET expression vector and introduced into Escherichia coli. Induction of protein expression with isopropyl beta-D-thiogalactopyranoside resulted in production of recombinant apoE3(1-183). Immunoblot analysis revealed that recombinant protein was present in both the cell pellet and cell culture supernatant. Analysis revealed that a significant portion of the rApoE3(1-183) in the cell pellet still possessed the bacterial N-terminal pel B leader sequence, encoded by plasmid DNA directly upstream of the apoE3(1-183) coding sequence. By contrast, this hydrophobic leader sequence had been removed from recombinant protein specifically accumulating in the culture medium. This behavior is novel for bacterial expression of apolipoprotein E and its truncated variants and permits efficient overexpression of the recombinant protein (> 100 mg/L cell culture). Recombinant apoE3(1-183) was isolated by a combination of heparin-Sepharose chromatography and reverse-phase HPLC. Electrospray mass spectrometry provided a mass of 21 191 daltons, corresponding directly to that expected from the known sequence. Circular dichroism spectroscopy revealed that the recombinant protein possesses significant amounts of alpha-helical secondary structure. The lipid binding ability of rApoE3(1-183) was evaluated using an in vitro lipoprotein binding assay. It was observed that recombinant apoE3(1-183) protected human low density lipoprotein (LDL) from lipid accumulation induced particle aggregation, indicating that it is capable of associating with lipoprotein surfaces. In addition, rApoE3(1-183) forms disk complexes with the model phospholipid dimyristoylphosphatidylcholine. In competition experiments, it was observed that rApoE3(1-183) phospholipid disks compete with 125I-LDL for binding to the apoB/E receptor on human skin fibroblasts to an extent similar to that observed for intact rApoE3. Taken together, these data show that recombinant apoE3(1-183) is fully functional as an apolipoprotein and receptor ligand. Given the high expression level and its known existence as a monomer in solution, we evaluated the potential for application of NMR spectroscopy to study the structure-function relationship of rApoE3(1-183). Bacteria were cultured in media supplemented with 15NH4Cl or [15N]glycine and the isotopically labeled recombinant apoE3(1-183) was analyzed by heteronuclear single quantum correlation NMR spectroscopy. The data revealed that rApoE3(1-183) is an excellent candidate for solution structure studies by NMR, including conformational adaptations associated with lipid association.
The flexibility of the polar side chains in the alpha-helical Type I antifreeze protein (AFP) near the solution freezing temperature was investigated by two-dimensional nuclear magnetic resonance spectroscopy. These experiments were conducted to define the rotameric conformations of the proposed ice-binding groups, threonines and asparagines, in order to probe the molecular mechanism for ice binding. On the basis of the 3J alpha beta 2 NMR coupling constant values of 7.1, 8.5, 8.5, and 6.8 Hz for residues T2, T13, T24, and T35, respectively, it can be calculated that the regularly spaced ice-binding threonines sample many possible rotameric states prior to ice binding. The lack of a dominant side chain rotamer is further corroborated by nuclear Overhauser distance measurements for T13 and T24. N16 and N27, both with 3J alpha beta 2 and 3J alpha beta 3 coupling constants of 8.4 and 4.5 Hz, respectively, show a slight preference for the side chain conformation with a chi 1 of -60 degrees. These data suggest that prior to ice binding the threonine and asparagine side chains are free to rotate and that a unique preformed ice-binding structure in solution is not apparent. These observations do not support the rigid side chain model proposed recently by an X-ray study [Sicheri, F., & Yang, D. S. C. (1995) Nature 375, 427-431].
The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.
In order to assess the importance of the buried Glu-38 observed in the structure of interleukin-8, an analog in which Glu-38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln-8 and Cys-9 amide proton chemical shifts, which are significantly downfield-shifted in the native protein, exhibit more 'normal' values. This observation indicates that in the native protein, Glu-38 side-chain carboxylate interacts with Gln-8 and Cys-9 amide protons. Although the N-terminal residues are critical for function, this interaction is not essential for neutrophil activation.
Desmopressin is a synthetic analog of the peptide hormone vasopressin in which the N-terminal alpha-amino group has been removed and L-arginine in position 8 has been replaced by D-arginine. Using 1H-NMR spectroscopy, we show that desmopressin binds to neurophysin-II, whereas deamino-vasopressin does not bind. Thus, the change in configuration at Arg8 causes a significant difference in the binding of these hormones to neurophysin-II. We have determined the structure of desmopressin bound to neurophysin-II using two-dimensional 1H nuclear magnetic resonance-transferred nuclear Overhauser effect techniques. A common binding motif for vasopressin and desmopressin is proposed that includes a positive charge group along with the hydrophobic surface formed by the side chains of Tyr2 and a beta-methylene provided by Phe-3. In vasopressin, the positive charge is provided by the N-terminal NH3+, whereas in desmopressin, the side chain of Arg-8 contributes the positive charge. The type II beta-turn found in residues Cys6-Pro7-D-Arg8-Gly9 of the bound structure of desmopressin folds the Arg8 side chain back toward the disulfide-bond loop, which allows the positive charged side chain of Arg8 to participate in binding. Such a type II beta-turn is not found in deamino-vasopressin in the presence of neurophysin-II.
BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.
1H-NMR spectroscopy and analytical ultracentrifugation studies reveal that monocyte chemoattractant protein-3 (MCP-3) is a monomer. NMR solution structure shows that MCP-3 adopts an alphabeta fold similar to what is observed in structures of other known chemokines. However, MCP-3 is unique in that it does not show a propensity to form dimers. The closely related chemokines MCP-1 and MCP-2 show a monomer-dimer equilibrium in sedimentation equilibrium studies (approximately 0.2-2 mg/ml). As these proteins are present at nanomolar concentrations in vivo, the results suggest that they are monomeric at functional concentrations and that the monomer is the functionally significant form of MCP-1, MCP-2 and MCP-3.
A series of lactam-bridged and linear 14 residue amphipathic alpha-helical peptides based on the sequence Ac-EXEALKKEXEALKK-amide were prepared in order to determine the effect of decreasing the hydrophobicity of the nonpolar face to helical content and stability. This was done by substituting position X by Ile, Val, and Ala. Lactam bridges spaced i to i + 4 were formed between the side chains of Glu3 and Lys7 and Glu10 and Lys14 while the linear noncyclized peptides could potentially form i to i + 4 salt bridges with the same residues. It was found that in all cases the lactam-bridged peptides were substantially more helical than the corresponding linear peptides as determined by CD spectroscopy. Moreover, the helical content approached 100% for the lactam-bridged peptides X = Ile and Ala and was greater than 80% for X = Val. For X = Ile and Val, this was partly due to the ability of the lactam bridges to enhance interchain interactions relative to the linear versions of the same sequence. Size-exclusion chromatography demonstrated that the Ile-based peptide associates as a dimer. The alanine-based lactam-bridged peptide was found to be monomeric as determined by concentration dependency studies and size-exclusion chromatography. Thermal denaturation studies in benign media indicated that the lactam-based peptides were very stable. The conformation of the Ala-based lactam peptide was further characterized by two-dimensional NMR spectroscopy and was found to be highly helical. The results demonstrate the ability of lactam bridges to stabilize the helical conformation and enhance dimerization of peptides based on a 3,4 hydrophobic heptad repeat. The substitution of Ala residues in the hydrophobic face of the alpha-helix can prevent dimerization and specify monomeric helical structure.
The C-terminal domain from the muscle protein troponin C (TnC) comprises two helix-loop-helix calcium-binding sites (residues 90-162). The assembly of these two sites is governed by calcium binding enabling a synthetic C-terminal domain to be preferentially and stoichiometrically assembled from two synthetic peptides (residues 93-126, SCIII, and 129-162, SCIV) in the presence of calcium only. It is therefore of great interest to know how closely the structure of this heterodimeric domain is to the intact protein domain. Analysis of such a structure has important implications in protein engineering and in understanding the stability of calcium-binding proteins in terms of biological function. The solution structure of this heterodimeric protein was determined by 1H NMR spectroscopy using 802 NOE derived distance restraints and 23 phi and 22 chi angle restraints. Distance geometry-simulated annealing calculations yielded a family of 42 converged structures (rmsd 0.86 +/- 0.17 A) showing an arrangement of four alpha-helices similar in fold to the C-terminal of troponin C. The dimer interface has several important interactions between helix pairs E/H and F/G responsible for the association of the two peptides. However, neither the peptide complex nor the solution NMR structure of TnC pack as tightly as that observed in the TnC X-ray structure. The interhelical distance between the F/G helix is about 1.4 A greater in solution than in the crystal. A comparison of the exposed surface area of the hydrophobic residues in the SCIII/SCIV heterodimer revealed that residues 1104, Y112, and 1121 are more exposed than in the previously determined solution structure of the SCIII homodimer. These residues are important for the interaction with the inhibitory region of TnI and provide evidence for their involvement in the regulation of muscle contraction.
The HPLC retention behavior of three complete single methionine and methionine sulfoxide replacement sets of two 18-mer model peptides and neuropeptide Y (NPY) were investigated. All peptides were prepared by multiple solid-phase peptide synthesis. Plotting the retention time differences between methionine and methionine sulfoxide analogues vs the position of replacement shows that potentially alpha-helical peptides become helical on binding during reversed-phase high performance liquid chromatography. In the case of an amphipathic alpha-helix, the retention time differences change periodically with a 3-4 repeat pattern, which allow the location of amphipathic helical structures. Replacements in nonamphipathic alpha-helical domains cause local preferential binding areas and lead to sequence-dependent retention time profiles. Methionine replacement studies of NPY suggest an unstructured or extended conformation from Tyr1 to Ala12 connected to a well-defined amphipathic alpha-helix from Pro13 to Arg35. The assignment is confirmed by comparison of nuclear Overhauser effects based two-dimensional 1H-nmr spectroscopy and utilization of the C alpha H shift index method in 50% trifluoroethanol/50% water.
2D 1H NMR spectroscopy of two alpha-helical peptides which differ in their amphipathicity has been used to investigate the relationships between amide-proton chemical shifts, amide-proton exchange rates, temperature, and trifluoroethanol (TFE) concentration. In 50% TFE, in which the peptides are maximally helical, the amide-proton chemical shift and temperature coefficient patterns are very similar to each other in each peptide. Temperature coefficients from -10 to -6 ppb/K, usually indicative of the lack of intramolecular hydrogen bonds, were observed even for hydrophobic amino acids in the center of the alpha-helices. However, slow hydrogen isotope exchange for residues from 4 to 16 in both 18-mer helices indicates intact intramolecular hydrogen bonds over most of the length of these peptides. Based on these anomalous observations, we suggest that the pattern of amide-proton shifts in alpha-helices in H20/TFE solvents is dominated by bifurcated intermolecular hydrogen-bond formation between the backbone carbonyl groups and TFE. The amide-proton chemical shift changes with increasing temperature may be interpreted by a disruption of intermolecular hydrogen bonds between carbonyl groups and the TFE in TFE/water rather than by the length of intramolecular hydrogen bonds in alpha-helices.
Mutation of residues at the ice-binding site of type III antifreeze protein (AFP) not only reduced antifreeze activity as indicated by the failure to halt ice crystal growth, but also altered ice crystal morphology to produce elongated hexagonal bipyramids. In general, the c axis to a axis ratio of the ice crystal increased from approximately 2 to over 10 with the severity of the mutation. It also increased during ice crystal growth upon serial dilution of the wild-type AFP. This is in marked contrast to the behavior of the alpha-helical type I AFPs, where neither dilution nor mutation of ice-binding residues increases the c:a axial ratio of the ice crystal above the standard 3.3. We suggest that the ice crystal morphology produced by type III AFP and its mutants can be accounted for by the protein binding to the prism faces of ice and operating by step growth inhibition. In this model a decrease in the affinity of the AFP for ice leads to filling in of individual steps at the prism surfaces, causing the ice crystals to grow with a longer c:a axial ratio.
BACKGROUND: The conformational preferences of a number of peptides with sequences related to the envelope glycoproteins of HIV-1 have been investigated in the past few years. Similar studies have not been made for HIV-2, which is a distinct virus with similar physiological effects to those of HIV-1. The discovery of common structural features would be a promising route to the design of immunogens for generally effective HIV vaccines. We present the results of an NMR conformational study of a sequence deriving from the V3 loop of HIV-2. RESULTS: Three synthetic immunogenic peptides were studied, of 12, 22 and 39 amino acids in length, all containing a central Met-Ser-Gly-Arg sequence conserved among a number of HIV-2 isolates. In addition, the 39-mer contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to comprise an important structural and immunological component of the intact glycoprotein. All three peptides display well defined beta-turns in the Met-Ser-Gly-Arg sequence, independent of the integrity of the disulfide bond. No other conformational preferences for folded conformations were found for the peptides. CONCLUSIONS: The presence of a beta-turn in the Met-Ser-Gly-Arg sequence is strikingly similar to the behavior seen for the corresponding principal neutralizing determinant sequence from gp120 of HIV-1 and argues, in the absence of information of the three-dimensional structure of the intact proteins, for a similarity in the structure of this region that could be exploited in the design of synthetic peptide vaccines generally effective against HIV infections.
The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.