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To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-1-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/M98L and V43A mutation effects are nonadditive. The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c.

Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c. The functionality assay is based upon the ability of yeast cells to grow in YPGE medium. Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue mutations, the I20 → V and M98 → L pair and the L9 → I and L85 → I pair. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for the role of those specific atom–atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.

This work is the first in a series devoted to applying mode coupling diffusion theory to the derivation of local dynamics properties of proteins in solution. The first-order mode-coupling approximation, or optimized Rouse–Zimm local dynamics (ORZLD), is applied here to derive the rotational dynamics of the bonds and compare the calculated with the experimental nmr ¹⁵N spin–lattice relaxation time behavior of the vnd/NK-2 homeodomain from Drosophila melanogaster. The starting point for the calculations is the experimental three-dimensional structure of the homeodomain determined by multidimensional nmr spectroscopy. The results of the computations are compared with experimentally measured ¹⁵N spin–lattice relaxation times T1, at 34.5 and 60.8 MHz, to check the first-order approximation. To estimate the relative importance of internal and overall rotation, both rigid and fluctuating dynamic models are examined, with fluctuations evaluated using molecular dynamics (MD) simulations. The correlation times for the fundamental bond vector time correlation function and for the second-order bond orientational TCF are obtained as a function of the residue number for vnd/NK-2. The stability of the corresponding local dynamics pattern for the fluctuating structure as a function of the length of the MD trajectory is presented. Diffusive dynamics, which is essentially free of model parameters even at first order in the mode-coupling diffusion approach, confirm that local dynamics of proteins can be described in terms of rotational diffusion of a fluctuating quasi-rigid structure. The comparison with the nmr data shows that the first-order mode coupling diffusion approximation accounts for the correct order of magnitude of the results and of important qualitative aspects of the data sensitive to conformational changes. Indications are obtained from this study to efficiently extend the theory to higher order in the mode-coupling expansion. These results demonstrate the promise of the mode-coupling approach, where the local dynamics of proteins is described in terms of rotational diffusion of a fluctuating quasi-rigid structure, to analyze nmr spin–lattice relaxation behavior. © 1999 John Wiley & Sons, Inc. Biopoly 49: 235–254, 1999

The three-dimensional solution structure obtained by NMR of the complex formed between the uniformly singly 15N and doubly 13C/15N-labeled vnd/NK-2 homeodomain and its consensus 16 base-pair DNA binding sequence was determined. This work was carried out using the accepted repertoire of experiments augmented with a novel implementation of the water flipback technique to enhance signals from exchangeable amide protons. The results using this new technique confirm the existence of hydrogen bonding between the invariant Asn51 and the second adenine of the DNA binding sequence, as seen in crystal structures of other homeodomain-DNA complexes, but never before detected by NMR. Hydrogen bonding by Arg5 and Lys3 in the minor groove of the DNA appears to be responsible for two unusually upfield-shifted ribose H1′ resonances. The DNA duplex is nearly straight and its structure is primarily that of B-DNA. A detailed comparison is presented for all available homeodomain-DNA structures including the vnd/NK-2 DNA complex, which demonstrates that homology is maintained in the protein structure, whereas for the orientation of the homeodomain relative to DNA, small but significant variations are observed. Interactions are described involving certain residues in specific positions of the homeodomain, namely Leu7, Thr41, and Gln50 of vnd/NK-2, where single amino acid residue mutations lead to dramatic developmental alterations. The availability of our previously determined three-dimensional structure of the vnd/NK-2 homeodomain in the absence of DNA allows us to assess structural changes in the homeodomain induced by DNA binding.

A novel implementation of the water flipback technique employing a 45° flip-angle water-selective pulse is presented. The use of this water flipback technique is shown to significantly enhance signal in 3D 15N-edited ROESY in a 20 kDa complex of the vnd/NK-2 homeodomain bound to DNA. The enhancement is seen relative to the same experiment using weak water presaturation during the recovery delay. This enhancement is observed for the signals from both labile and nonlabile protons. ROESY and NOESY pulse sequences with 45° water flipback are presented using both HMQC and HSQC for the 15N dimension. The 45° flipback pulse is followed by a gradient, a water selective 180° pulse, and another gradient to remove quadrature images and crosspeak phase distortion near the water frequency. Radiation damping of the water magnetization during the t1and t2evolution periods is suppressed using gradients. Water resonance planes from NOESY–HMQC and NOESY–HSQC spectra show that the HMQC version of the pulse sequences can provide stronger signal for very fast exchanging protons. The HSQC versions of the ROESY and NOESY pulse sequences are designed for the quantitative determination of protein–water crossrelaxation rates, with no water-selective pulses during the mixing time and with phase cycling and other measures for reducing axial artifacts in the water signal.

An ‘antisense’ peptide (‘HTCA’), whose sequence was generated by reading the antisense RNA sequence corresponding to ACTH(1–24) was shown to bind ACTH(1–24) with a Kdof 0.3 nM in a solid‐matrix binding assay [(1986) Biochem. J. 234, 679–683]. Two‐dimensional NMR spectra were used to examine the conformational behavior in methanol and in water solution of two fragments of adrenocorticotropin, ACTH(1–24) and ACTH(1–13), as well as their antisense peptides, HTCA and HTCA(12–24). The conformations are extended chains in these solutions, both as isolated molecules and when mixed with their antisense complements. The Kdvalues are greater than 1 mM.

The lipid-water interface is critical for the packing of lipid molecules in membranes. We have demonstrated that lateral phase separation in membranes can be driven by electrostatic interactions such as those involving charged lipid species and oppositely charged peptides, in addition to hydration effects at the lipid-water interface. By using nuclear magnetic resonance (NMR), circular dichroism and fluorescence spectroscopy we have shown that binding of a 21-amino acid peptide containing six positively charged arginine residues to mixed phosphatidylcholine (PC)/phosphatidylglycerol (PG) membranes results in a conformational change in the peptide from a random coil to a helical structure and causes the formation of domains of negatively charged PG. Binding of the peptide to PG membranes disorders the lipid hydrocarbon chains. The strength of lipid-peptide binding at the interface, the conformational change in the peptide, and domain formation with the negatively charged lipid are coupled energetically. The lipid-peptide association constant is lower for membranes containing 20 mol% PG in PC/PG mixtures than for 100% PG membranes. We suggest that one of the factors that lower the association constant in PC/PG membranes is entropic energy of formation of PG domains. Besides electrostatic interactions, hydration of lipids is important for domain formation. We have shown that dipalmitoylphosphatidylcholine and dipalmitoylphosphatidyl-ethanolamine separate under conditions of decreased water activity. Furthermore, water activity controls lipid packing stress in the hydrocarbon core and the headgroups of membranes as demonstrated by induction of an inverse-hexagonal-to-lamellar phase transition in dioleoylphosphatidylethanolamine. The experiments have shown that lipid-peptide and lipid-water interactions at the interface influence the packing of lipid hydrocarbon chains. Consequently we predict that a change in lipid-lipid interaction in the hydrocarbon core of the membrane, for example as a result of the introduction of polyunsaturated fatty acids, will alter lipid-solvent and lipid-peptide interactions at the interface.

Binding of the peptide fragment 828-84. (P828), amino acid sequence RVIEVVQGACRAIRHIPRRIR, from the carboxy-terminal region of the envelope glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) to membranes composed of a mixture of neutral and negatively charged phospholipids results in domain or cluster formation of the charged lipid. The conformation and dynamics of the peptide are investigated in solution and in the presence of sodium dodecyl sulphate (SDS) micelles using high resolution nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectropolarimetry. The CD results demonstrate that addition of either SDS, negatively charged phospholipid liposomes, or trifluoroethanol (TFE) induces a conformational transition of the peptide from a random coil or an extended chain in water to a more ordered structure with an estimated helical content of up to 60%. The structure of the peptide in a membrane mimetic SDS solution was investigated in detail using two-dimensional NMR. The measurements demonstrate the existence of a helical component in the peptide conformation in the SDS-bound state. The peptide most likely exists as an ensemble of conformations with exchange times between them which are fast on the chemical shift NMR time scale (10-3 s). Simple neutralization of the six arginine sidechain charges does not cause the peptide to adopt an ordered structure. Thus, there is an additional requirement for the structural transition such as that resulting from constraint of the peptide on a surface, or localization of the peptide at the lipid-water interface where the polarity is lower. Our results favour a model of the peptide-lipid interaction in which the peptide backbone is located in the water phase and part of the amino acid sidechains penetrate the lipid-water interface.

We describe the NMR determination of the three-dimensional structure of a 77 amino acid residue protein, which consists of the 60 residue NK-2 homeodomain fromDrosophila melanogasterand adjacent amino acid residues. The NK-2 homeodomain protein is part of a 723 amino acid residue protein which is expressed early in embryonic development in part of the central nervous system. NK-2 was characterized using both a natural abundance and a uniformly¹⁵N enriched sample by two-dimensional and three-dimensional NMR experiments. The average root-mean-square deviation for 30 structures for residues 8 to 53 is 0.40 Å for the backbone heavy-atoms and 0.72 Å for the backbone and side-chain heavy-atoms. These structures were obtained from 986 NOE-derived upper and lower bound restraints. The three-dimensional structure contains three helices which consist of homeodomain amino acid residues 10 to 22, 28 to 38 and 42 to 52, as well as a turn between helix II and III, characteristic of homeodomains. Residues 53 to 60 of the DNA recognition helix are not fully ordered in the absence of DNA. In the free state this segment adopts a flexible but helix-like structure between residues 53 and 56 and is disordered from residues 57 to 60 although, as shown previously, the helix elongates by eight residues upon binding to DNA. The role of variable residues 52, 54 and 56 in determining the structure and flexibility of the recognition helix, as well as the stability of the NK-2 homeodomain as manifested by its thermal denaturation, are discussed.

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N-methylation of the third amino acid residue in the sequence and the negatively charged N-terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded β-pleated sheet. The observed senktide-receptor specificity is suggested to result from a selection of this or a closely related conformation.

Two pulse sequences are described that employ a modified water flipback technique to enhance the signal intensity of weak side chain resonances at the protein–DNA interface of the vnd/NK-2 homeodomain/DNA complex in an15N-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of the NOESY mixing time, optimizing the direct NOE transfer of magnetization from the water to the protein by maximizing thez-component of the water magnetization. In one of the pulse sequences, radiation damping during the the indirect1H and15N evolution times is suppressed. A modified version of the WATERGATE water suppression technique is employed during the HMQC portion of the experiment. The signal enhancement is demonstrated for the resonances of the side chain amide of Asn51, an invariant homeodomain residue whose contact with the DNA is critical for binding. An ancillary advantage of the experiment is the ability to observe NOE transfer of magnetization from water. The information present in the water resonance plane of the three-dimensional spectrum is illustrated in a comparison with the corresponding HMQC spectrum of the protein/DNA complex.

An ‘antisense’ peptide (‘HTCA’), whose sequence was generated by reading the antisense RNA sequence corresponding to ACTH(1–24) was shown to bind ACTH(1–24) with a Kdof 0.3 nM in a solid-matrix binding assay [(1986) Biochem. J. 234, 679–683]. Two-dimensional NMR spectra were used to examine the conformational behavior in methanol and in water solution of two fragments of adrenocorticotropin, ACTH(1–24) and ACTH(1–13), as well as their antisense peptides, HTCA and HTCA(12–24). The conformations are extended chains in these solutions, both as isolated molecules and when mixed with their antisense complements. The Kdvalues are greater than 1 mM.

A point mutation (Ala-589 to Thr) in the transmembrane protein of the human immunodeficiency virus type 1 (HIV-1) has been shown to decrease the sensitivity of the virus to the neutralizing effect of human HIV-1 specific antibodies [(1990) J. Virol. 64, 3240-3248]. Here 17-residue peptides with the parental and mutant sequences were compared: the parental peptide bound antibodies of sera from HIV-1 infected persons more frequently and with higher affinity than the mutant peptide. However, according to circular dichroism (CD), NMR spectroscopy and molecular modelling the peptides have indistinguishable backbone conformations under a variety of experimental conditions. These techniques showed for both peptides that no ordered helix was present in water solution. However, for both peptides in alcohol-water solutions approximately 60% α-helix coula be induced. The three-dimensional structures of these peptides provide a basis for understanding how this mutation in the transmembrane protein may affect the interaction with both the outer envelope glycoprotein and with antibodies.

Secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2gene from Drosophila melanogasterare described. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles in structure and sequence specificity of individual atom-atom interactions are described.

A point mutation (Ala‐589 to Thr) in the transmembrane protein of the human immunodeficiency virus type 1 (HIV‐1) has been shown to decrease the sensitivity of the virus to the neutralizing effect of human HIV‐1 specific antibodies [(1990) J. Virol. 64, 3240‐3248]. Here 17‐residue peptides with the parental and mutant sequences were compared: the parental peptide bound antibodies of sera from HIV‐1 infected persons more frequently and with higher affinity than the mutant peptide. However, according to circular dichroism (CD), NMR spectroscopy and molecular modelling the peptides have indistinguishable backbone conformations under a variety of experimental conditions. These techniques showed for both peptides that no ordered helix was present in water solution. However, for both peptides in alcohol‐water solutions approximately 60% α‐helix coula be induced. The three‐dimensional structures of these peptides provide a basis for understanding how this mutation in the transmembrane protein may affect the interaction with both the outer envelope glycoprotein and with antibodies.

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N‐methylation of the third amino acid residue in the sequence and the negatively charged N‐terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded β‐pleated sheet. The observed senktide‐receptor specificity is suggested to result from a selection of this or a closely related conformation.

Chromogranin A is known to undergo pH induced conformational changes, and the difference in conformation is supposed to be responsible for the difference in Ca2+binding property. To gain insight regarding the overall structure and the nature of pH‐induced conformational changes of chromogranin A, limited trypsin digestions were carried out at pH 5.5 and pH 7.5. The resulting fragments were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and the amino acid sequences of the tryptic fragments were determined. From these analyses it was shown that the chromogranin A structure consists of an N‐terminal compact core region and a rather loosely organized C‐terminal region and that the change of pH from 7.5 to 5.5 loosened the overall structure of chromogranin A, exposing the C‐terminal region. Since the conserved C‐terminal region (residues 407–431) was shown to exist in monomer‐dimer and monomer‐tetramer equilibria at pH 7.5 and 5.5, respectively, the conformational changes of the region at pH 7.5 and 5.5 were studied by circular dichroism spectroscopy using a synthetic peptide representing the conserved C‐terminal region. When the pH was changed from 7.5 to 5.5, the coil structure of the C‐terminal peptide decreased with an accompanying increase of α‐helicity.