Biomedical subjects

B D Sykes

Publications and source records attributed to B D Sykes.

At least 55 records · Page 3Linked to original sources

PubMed1998

The ice-binding site of sea raven antifreeze protein is distinct from the carbohydrate-binding site of the homologous C-type lectin.

Antifreeze proteins lower the freezing point of their solution by binding to ice and inhibiting its growth. One of several structurally different antifreeze proteins in fishes (type II) is homologous to the carbohydrate-recognition domain of Ca2+-dependent lectins and adopts the same three-dimensional fold. Type II antifreeze proteins from herring and smelt require Ca2+ for binding to ice, whereas this same antifreeze protein in sea raven binds to ice in the absence of Ca2+ and has only two of the five Ca2+-liganding amino acids that are present in the lectin. To locate the ice-binding site, site-directed mutants of the 15 kDa, globular, disulfide-bonded sea raven antifreeze protein were produced by secretion from Pichia pastoris. Pairs of amino acid replacements, insertions, and a peptide loop swap were made in the region equivalent to the sugar-binding site of the lectin that encompasses loops 3 and 4 and beta-sheets 7 and 8. Even the most extensive mutation caused only a 25% decrease in antifreeze activity and demonstrated that the residues corresponding to the Ca2+-binding site are only peripherally involved in ice binding. When adjacent surface residues were mutated, the replacement of one residue, Ser120 by His, caused a 35% decrease in activity by itself and an 80% loss in conjunction with the peptide loop swap mutation. This pivotal sea raven antifreeze protein amino acid does not coincide with the herring ice-binding epicenter, but is located within the region corresponding to the proposed CaCO3-binding surface of a third homologue, the pancreatic stone protein. Intron and exon structure of the sea raven AFP gene also suggests that it might be more closely related to the stone protein gene than to the lectin gene. These results support the notion that this family of proteins has evolved more than one binding surface from the same protein scaffold.

Animals↗

PubMed1998

Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein.

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10-30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive beta structure (70-80%) with little or no alpha helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0 degrees C to 20 degrees C), and the presence of 50% trifluoroethanol.

Amino Acid Sequence↗

PubMed1998

Structure-based minimization of transforming growth factor-alpha (TGF-alpha) through NMR analysis of the receptor-bound ligand. Design, solution structure, and activity of TGF-alpha 8-50.

The investigation of a N-terminally truncated human transforming growth factor-alpha (TGF-alpha; residues 8-50) has been completed to determine the contribution of the N terminus to receptor binding and activation. The deletion protein was proposed and designed through study of NMR relaxation and nuclear Overhauser enhancement data obtained from the TGF-alpha-epidermal growth factor (EGF) receptor complex, which indicated that the residues N-terminal to the A loop remain flexible in receptor-bound TGF-alpha and thus suggested their lack of involvement in receptor binding (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292; McInnes, C., Hoyt, D. W., Harkins, R. N., Pagila, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1996) J. Biol. Chem. 271, 32204-32211). TGF-alpha 8-50 was shown to have approximately 10-fold lower affinity for the receptor than the native molecule in an assay quantifying the ability to compete with EGF for binding and to have a similar reduction in activity as indicated by a cell proliferation assay. NMR solution structural calculations on this molecule demonstrate correct formation of the three disulfide bonds of TGF-alpha 8-50 and have established the presence of native secondary structure in the B and C loops of the protein. However, some perturbation of the global fold with respect to the orientation of the subdomains was observed. These results suggest that although the N-terminal residues do not contribute directly to binding, they make a significant contribution in defining the conformation of the growth factor, which is required for complete binding and activity and is therefore significant in terms of producing native folding of TGF-alpha. They also show that information obtained from the receptor-bound ligand can be used to guide the design and minimization of TGF-alpha analogues. The implications of the study of TGF-alpha 8-50 for the design and synthesis of reductants of this growth factor are therefore discussed.

Amino Acid Sequence↗

PubMed1998

Structure and interaction site of the regulatory domain of troponin-C when complexed with the 96-148 region of troponin-I.

The structure of the regulatory domain of chicken skeletal troponin-C (residues 1-90) when complexed with the major inhibitory region (residues 96-148) of chicken skeletal troponin-I was determined using multinuclear, multidimensional NMR spectroscopy. This complex represents the first interaction formed between the regulatory domain of troponin-C and troponin-I after calcium binding in the regulation of muscle contraction. The stoichiometry of the complex was determined to be 1:1, with a dissociation constant in the 1-40 microM range. The structure of troponin-C in the complex was calculated from 1039 NMR distance and 111 dihedral angle restraints. When compared to the structure of this domain in the calcium saturated "open" form but in the absence of troponin-I, the bound structure appears to be slightly more "closed". The troponin-I peptide-binding site was found to be in the hydrophobic pocket of calcium saturated troponin-C, using edited/filtered NMR experiments and chemical shift mapping of changes induced in the regulatory domain upon peptide binding. The troponin-I peptide (residues 96-148) was found to bind to the regulatory domain of troponin-C very similarly, but not identically, to a shorter troponin-I peptide (region 115-131) thought to represent the major interaction site of troponin-I for this domain of troponin-C.

Animals↗

PubMed1998

Solution structure of eotaxin, a chemokine that selectively recruits eosinophils in allergic inflammation.

The solution structure of the CCR3-specific chemokine, eotaxin, has been determined by NMR spectroscopy. The quaternary structure of eotaxin was investigated by ultracentrifugation and NMR, and it was found to be in equilibrium between monomer and dimer under a wide range of conditions. At pH </= 5 and low ionic strength, eotaxin was found to be predominantly a monomer. The three-dimensional structure of the eotaxin monomer solved at pH 5.0 revealed that it has a typical chemokine fold, which includes a 3-stranded beta-sheet and an overlying alpha-helix. Except for the N-terminal residues (residues 1-8), the core of the protein is well defined. The eotaxin structure is compared with the chemokines regulated upon activation, normal T-cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1); eotaxin binds only CC chemokine receptor CCR3, whereas RANTES binds many receptors including CCR3, and MCP-1 binds a distinct receptor, CCR2. The RMSD of the eotaxin ensemble of structures with the RANTES average minimized monomeric subunit is 5.52 +/- 0.87 A over all backbone atoms and 1.14 +/- 0.09 A over backbone atoms of residues 11-28 and 34-65. The most important difference between the structures is in the N-terminal residues that are unstructured in eotaxin but structured in RANTES and MCP-1. Several residues in the loop region of RANTES show similar packing in eotaxin (residues 11-17). As the N-terminal and loop regions have been shown to be critical for receptor binding and signaling, this structure will be useful for determining the basis for CCR3 selectivity of the eotaxin.

Amino Acid Sequence↗

PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗

PubMed1998

Insights into the mechanism of heterodimerization from the 1H-NMR solution structure of the c-Myc-Max heterodimeric leucine zipper.

The oncoprotein c-Myc (a member of the helix-loop-helix-leucine zipper (b-HLH-LZ) family of transcription factors) must heterodimerize with the b-HLH-LZ Max protein to bind DNA and activate transcription. It has been shown that the LZ domains of the c-Myc and Max proteins specifically form a heterodimeric LZ at 20 degreesC and neutral pH. This suggests that the LZ domains of the c-Myc and Max proteins are playing an important role in the heterodimerization of the corresponding gene products in vivo. Initially, to gain an insight into the energetics of heterodimerization, we studied the stability of N-terminal disulfide-linked versions of the c-Myc and Max homodimeric LZs and c-Myc-Max heterodimeric LZ by fitting the temperature-induced denaturation curves monitored by circular dichroism spectroscopy. The c-Myc LZ does not homodimerize (as previously reported) and the c-Myc-Max heterodimeric LZ is more stable than the Max homodimeric LZ at 20 degreesC and pH 7.0. In order to determine the critical interhelical interactions responsible for the molecular recognition between the c-Myc and Max LZs, the solution structure of the disulfide-linked c-Myc-Max heterodimeric LZ was solved by two-dimensional 1H-NMR techniques at 25 degreesC and pH 4.7. Both LZs are alpha-helical and the tertiary structure depicts the typical left-handed super-helical twist of a two-stranded parallel alpha-helical coiled-coil. A buried salt bridge involving a histidine on the Max LZ and two glutamate residues on the c-Myc LZ is observed at the interface of the heterodimeric LZ. A buried H-bond between an asparagine side-chain and a backbone carbonyl is also observed. Moreover, evidence for e-g interhelical salt bridges is reported. These specific interactions give insights into the preferential heterodimerization process of the two LZs. The low stabilities of the Max homodimeric LZ and the c-Myc-Max heterodimeric LZ as well as the specific interactions observed are discussed with regard to regulation of transcription in this family of transcription factors.

Amino Acid Sequence↗

PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

Databases as Topic↗

PubMed1998

Recombinant locust apolipophorin III: characterization and NMR spectroscopy.

Apolipophorin III (apoLp-III) from the locust Locusta migratoria is an exchangeable apolipoprotein that reversibly binds to lipoproteins. During lipid binding the protein has been proposed to undergo a major conformational change. To study the mechanism of lipid binding we have cloned and expressed recombinant protein in bacteria, permitting stable isotope enrichment for heteronuclear NMR spectroscopy and site-directed mutagenesis. The cDNA coding for apoLp-III was subcloned into the pET expression vector and transformed into Escherichia coli cells. Induction of expression resulted in the specific appearance of apoLp-III in the cell culture medium, indicating it escaped the bacteria without lysis. The protein was purified from the cell-free supernatant by reversed-phase HPLC, characterized and compared to the natural protein isolated from locust hemolymph. SDS-PAGE revealed the recombinant protein has a molecular mass of approximately 17 kDa, similar to that of deglycosylated natural apoLp-III. Monoclonal antibodies were used to detect recombinant apoLp-III in the cells as well as in cell-free medium of induced bacterial cultures. Amino acid sequencing and analysis confirmed the identity of the recombinant protein as L. migratoria apoLp-III. Circular dichroism spectroscopy of recombinant and natural apoLp-III showed similar spectra, both displaying high contents of alpha-helical secondary structure. Denaturation studies of lipid-free apoLp-III with guanidine hydrochloride showed that both proteins have similar denaturation midpoints and DeltaG values indicating similar protein stability. The natural and recombinant protein were functional in lipoprotein binding assays. Using recombinant protein, uniformly and specifically labeled with 15N-amino acids, two dimensional 1H-15N heteronuclear single quantum correlation spectra were obtained. The spectra revealed excellent chemical shift dispersion in both the 1H and 15N dimensions with a well defined resonance pattern. Studies with 15N-leucine specifically labeled apoLp-III in the presence and absence of the micelle forming lipid, dodecylphosphocholine, provided evidence for a significant conformational change upon lipid association.

Animals↗

PubMed1998

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity.

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

Animals↗

PubMed1998

Binding of an oligopeptide to a specific plane of ice.

The alpha-helical antifreeze protein (AFP) from winter flounder inhibits ice growth by binding to a specific set of pyramidal surface planes that are not otherwise macroscopically expressed. The 37-residue AFP contains three 11-amino acid repeats that make a stereo-specific fit to the ice lattice along the <01-12> direction of the (20-21) and equivalent binding planes. When the AFP was shortened to delete two of the three 11-amino acid ice-binding repeats, the resulting 15-residue peptide and its variants were less helical and showed no antifreeze activity. However, when the helicity of the peptide was reinforced by an internal lactam bridge between Glu-7 and Lys-11, the minimized AFP was able to stably express the pyramidal plane (20-21) on the surface of growing ice crystals. This dynamic shaping of the ice surface by a single ice-binding repeat provides evidence that AFP adsorption to the ice lattice is not an "all-or-nothing" interaction. Instead, a partial interaction can help develop the binding site on ice to which the remainder of the AFP (or other AFP molecules) can orient and bind.

Antifreeze Proteins↗

PubMed1998

The solution structure of type II antifreeze protein reveals a new member of the lectin family.

A recombinant form of the sea raven type II antifreeze protein (SRAFP) has been produced using the Pichia pastoris expression system. The antifreeze activity of recombinant SRAFP is indistinguishable from that of the wild-type protein. The global fold of SRAFP has been determined by two-dimensional 1H homonuclear and three-dimensional 1H-¿15N¿ heteronuclear NMR spectroscopy using 785 NOE distance restraints and 47 angular restraints. The molecule folds into one globular domain that consists of two helices and nine beta-strands in two beta-sheets. The structure confirms the proposed existence of five disulfide bonds. The global fold of SRAFP is homologous to C-type lectins and pancreatic stone proteins, even though the sequence identity is only approximately 20%.

Amino Acid Sequence↗

PubMed1998

Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy.

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.

Affinity Labels↗

PubMed1998

Interhelical contacts are required for the helix bundle fold of apolipophorin III and its ability to interact with lipoproteins.

Apolipophorin-III (apoLp-III) from the insect, Manduca sexta, is a 166-residue exchangeable apolipoprotein that plays a critical role in the dynamics of plasma lipoprotein interconversions. Our previous work indicated that a 36-residue C-terminal peptide fragment, generated by cyanogen bromide digestion of apoLp-III, was unable to bind to lipid surfaces (Narayanaswami V, Kay CM, Oikawa K, Ryan RO, 1994, Biochemistry 33:13312-13320), and showed no secondary structure in aqueous solution. In this paper, we have performed structural studies of this peptide (E131-Q166) complexed with SDS detergent micelles, or in the presence of the helix-inducing solvent trifluoroethanol (TFE), by two-dimensional 1H NMR spectroscopy. The peptide adopts an alpha-helical structure in the presence of both SDS and 50% TFE. The lipid-bound structure of the peptide, generated from the NMR NOE data, showed an elongated, slightly curved alpha-helix. Despite its high alpha-helix forming propensity, the peptide requires alpha helix-promoting environment to adopt an alpha-helical structure. This indicates the importance of the surrounding chemical environment and implies that, in the absence of lipid, tertiary contacts in the folded protein play a role in maintaining its structural integrity. Furthermore, the data suggest that the amphipathic helix bundle organization serves as a prerequisite structural motif for the reversible lipoprotein-binding activity of M. sexta apoLp-III.

Amino Acid Sequence↗

PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy.

Amino Acid Sequence↗

PubMed1998

CAMRA: chemical shift based computer aided protein NMR assignments.

A suite of programs called CAMRA (Computer Aided Magnetic Resonance Assignment) has been developed for computer assisted residue-specific assignments of proteins. CAMRA consists of three units: ORB, CAPTURE and PROCESS. ORB predicts NMR chemical shifts for unassigned proteins using a chemical shift database of previously assigned homologous proteins supplemented by a statistically derived chemical shift database in which the shifts are categorized according to their residue, atom and secondary structure type. CAPTURE generates a list of valid peaks from NMR spectra by filtering out noise peaks and other artifacts and then separating the derived peak list into distinct spin systems. PROCESS combines the chemical shift predictions from ORB with the spin systems identified by CAPTURE to obtain residue specific assignments. PROCESS ranks the top choices for an assignment along with scores and confidence values. In contrast to other auto-assignment programs, CAMRA does not use any connectivity information but instead is based solely on matching predicted shifts with observed spin systems. As such, CAMRA represents a new and unique approach for the assignment of protein NMR spectra. CAMRA will be particularly useful in conjunction with other assignment methods and under special circumstances, such as the assignment of flexible regions in proteins where sufficient NOE information is generally not available. CAMRA was tested on two medium-sized proteins belonging to the chemokine family. It was found to be effective in predicting the assignment providing a database of previously assigned proteins with at least 30% sequence identity is available. CAMRA is versatile and can be used to include and evaluate heteronuclear and three-dimensional experiments.

Amino Acid Sequence↗

PubMed1998

Unusual beta-sheet periodicity in small cyclic peptides.

Cyclic peptide homologs of gramicidin S containing 6, 8, 10, 12, 14 and 16 residues were synthesized and characterized using circular dichroism (CD) and 1H NMR spectroscopy. Based on the three-dimensional structures generated from these data we have found strong evidence of a periodic sequence-length dependence on beta-sheet content. In particular, peptides of length 6, 10 and 14 residues exhibit a high beta-sheet content, while peptides of 8, 12 and 16 residues appear to exist as random coils. This unusual beta-sheet periodicity may have important implications in our understanding of beta-sheet formation and in the design of constrained beta-sheet and beta-hairpin mimics.

Amino Acid Sequence↗

PubMed1998

NMR evidence for a conformational adaptation of apolipophorin III upon lipid association.

A characteristic property of amphipathic exchangeable apolipoproteins is an ability to exist alternately in lipid-free and lipid-bound states. In the present study, we have used 1H-15N-heteronuclear single quantum correlation spectroscopy to probe structural changes of apolipophorin III upon lipid association, by monitoring alterations of the chemical shifts of specific amino acids as a function of lipid titration. 15N-valine-, 15N-leucine-, 15N-lysine-, and 15N-glycine-labeled apolipophorin III were used in titration experiments with the micelle-forming lipid dodecylphosphocholine. In the absence of lipid, valine and leucine residues are located in the hydrophobic interior of the apolipophorin III helix bundle and their resonances resist chemical shift changes below the critical micelle concentration of dodecylphosphocholine. At the critical micelle concentration, however, dramatic and abrupt chemical shift changes occur, apparently coincident with formation of a protein-lipid micelle complex, as judged by significant line-width broadening of the crosspeaks. By contrast, apolipophorin III lysine and glycine residues are located on the hydrophilic surfaces of amphipathic alpha-helices or in loop regions, exposed to solvent. Their crosspeaks display either a chemical shift change similar to that seen for hydrophobic residues or a more gradual chemical shift change, beginning at very low dodecylphosphocholine concentrations. These results indicate that an interaction occurs between specific solvent-exposed lysine residues and dodecylphosphocholine below the critical micelle concentration of this lipid, whereas valine and leucine residues are not accessible to monomeric dodecylphosphocholine. At the critical micelle concentration, however, the availability of a newly formed lipid surface induces apolipophorin III binding, concomitant with conformational opening of the helix bundle, exposing its hydrophobic surfaces for binding to the dodecylphosphocholine micellar surface. Subsequently, hydrophobic residues undergo characteristic spectral changes. Subtle differences in behavior of specific hydrophobic residues, in terms of their response to dodecylphosphocholine titration and relative locations in the helix-bundle conformation, suggest that one end of the molecule may initiate contact with the lipid surface, followed by helix bundle opening.

Amino Acids↗