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F G Prendergast

Publications and source records attributed to F G Prendergast.

At least 37 records · Page 2Linked to original sources
PubMed1998

Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array.

Atomic level structures have been determined for the soluble forms of several colicins and toxins, but the structural changes that occur after membrane binding have not been well characterized. Changes occurring in the transition from the soluble to membrane-bound state of the C-terminal 190-residue channel polypeptide of colicin E1 (P190) bound to anionic membranes are described. In the membrane-bound state, the alpha-helical content increases from 60-64% to 80-90%, with a concomitant increase in the average length of the helical segments from 12 to 16 or 17 residues, close to the length required to span the membrane bilayer in the open channel state. The average distance between helical segments is increased and interhelix interactions are weakened, as shown by a major loss of tertiary structure interactions, decreased efficiency of fluorescence resonance energy transfer from an energy donor on helix V of P190 to an acceptor on helix IX, and decreased resonance energy transfer at higher temperatures, not observed in soluble P190, implying freedom of motion of helical segments. Weaker interactions are also shown by a calorimetric thermal transition of low cooperativity, and the extended nature of the helical array is shown by a 3- to 4-fold increase in the average area subtended per molecule to 4,200 A2 on the membrane surface. The latter, with analysis of the heat capacity changes, implies the absence of a developed hydrophobic core in the membrane-bound P190. The membrane interfacial layer thus serves to promote formation of a highly helical extended two-dimensional flexible net. The properties of the membrane-bound state of the colicin channel domain (i.e., hydrophobic anchor, lengthened and loosely coupled alpha-helices, and close association with the membrane interfacial layer) are plausible structural features for the state that is a prerequisite for voltage gating, formation of transmembrane helices, and channel opening.

Calorimetry, Differential Scanning↗
PubMed1998

Comparison of 15N- and 13C-determined parameters of mobility in melittin.

Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by 15N and 13C NMR. Specifically, inverse-detected 15N T1 and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 degrees C enriched with 15N at six amide positions and in the Trp19 side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and alpha-helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: tau m, the correlation time for the overall rotation; S2, a site-specific order parameter which is a measure of the amplitude of the internal motion; and tau e, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the 15N measurements and from 13C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678-1688]. tau m and tau e values were consistent from data on the two nuclei. In the MLT monomer, S2 values for the backbone N-H and C alpha-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C alpha-H order parameters. The Trp side-chain N-H and C-H order parameters, and tau e values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.

Amino Acid Sequence↗
PubMed1997

Water (H2O and D2O) molar absorptivity in the 1000-4000 cm-1 range and quantitative infrared spectroscopy of aqueous solutions.

Water (H2O and D2O) molar absorptivity was measured by Fourier transform infrared transmission spectroscopy in the 1000-4000 cm-1 range at 25 degrees C. A series of assembled cells with path lengths from 1.2 to 120.5 microns was used for these measurements. The optimal path length (the path length of aqueous solution at which the IR spectrum of solute, corrected for water absorbance, has the highest signal-to-noise ratio) was calculated for all water absorbance bands. The results presented here show that the optimal path length does not depend on solute properties and is inversely proportional to the solvent (water) molar absorptivity. The maximal signal-to-noise ratio for measurements of IR spectra of aqueous solution in the 1650 cm-1 spectral region, of primary interest in biological applications, can be obtained at an optimal cell path lengths of 3-4 microns (H2O) and 40-60 microns (D2O). As an example, the signal-to-noise ratio was calculated as a function of the cell path length for the amide I (H2O) and amide I' (D2O) bands of an aqueous lysozyme solution. The molar absorptivities of water bands are several orders of magnitude weaker than those of the strongest bands of biological macromolecules in the same spectral regions. High net water absorbance in aqueous solutions is due simply to the very high molar concentration of water. A method is proposed for the quantitative measuring of the path length of the cell which exploits the molar absorptivity of the strongest water bands (stretching vibrations) or of bands which do not overlap with solute absorbance. A path length in the range from approximately 0.01 micron to approximately 1.0 mm can be determined with high precision using this technique for a samples of known concentration. Problems involved in the proper correction of strong water absorbance in IR spectra of aqueous solutions of biomolecules are discussed, including multiple reflections within the cell, the effects of pH, temperature, and perturbation of water spectral properties by polar solutes, as well as the selection of optimal spectral regions in which one may obtain the most precise absorbance corrections.

Calcium Fluoride↗
PubMed1997

Main chain and side chain dynamics of peptides in liquid solution from 13C NMR: melittin as a model peptide.

Peptide backbone and lysine and tryptophan side chain mobilities in the synthetic, 26-residue peptide melittin (MLT) enriched with 13C were investigated in liquid solution by 13C T1 and steady state nuclear Overhauser effect measurements at two magnetic fields and by Trp fluorescence anisotropy measurements and were analyzed using the Lipari and Szabo model-free approach. The overall rotational correlation times at 20 degrees C were 1.28, 1.4, 2.8, and 4.2 ns for monomeric random coil MLT, for monomeric helical MLT (in CD3OD), for tetrameric MLT in neat D2O, and for the tetramer in 50 mM phosphate buffer, respectively. Motion of the backbone in the interior of the sequence was most restricted in the monomeric helix and least restricted in the tetramer. In the monomeric disordered peptide, relatively less restricted backbone motion extending from the N terminus to the fourth residue was observed. Such "end effects" continued only to the third residue in the monomeric helix and were observed just in the amino terminus glycine in the tetramer. The three Lys side chains showed the least restricted motion in the monomers and a differential restriction in the tetramers consistent with the tetramer structure. The motion of the Trp side chain was more restricted than that of Lys side chains and generally as restricted as that of the interior backbone atoms. The effective correlation times for the local motion of the backbone atoms were in the motional narrowing limit and showed distinct patterns. Agreement between NMR relaxation and Trp fluorescence anisotropy data was good for the monomer but not for the tetramer. Implications of these results for peptide dynamics in general are examined.

Amino Acid Sequence↗
PubMed1997

Photoacoustic analysis of proteins: volumetric signals and fluorescence quantum yields.

A series of proteins has been examined using time-resolved, pulsed-laser volumetric photoacoustic spectroscopy. Photoacoustic waveforms were collected to measure heat release for calculation of fluorescence quantum yields, and to explore the possibility of photoinduced nonthermal volume changes occurring in these protein samples. The proteins studied were the green fluorescent protein (GFP); intestinal fatty acid binding protein (IFABP), and adipocyte lipid-binding protein (ALBP), each labeled noncovalently with 1-anilinonaphthalene-8-sulfonate (1,8-ANS) and covalently with 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan); and acrylodan-labeled IFABP and ALBP with added oleic acid. Of this group of proteins, only the ALBP labeled with 1,8-ANS showed significant nonthermal volume changes at the beta = 0 temperature (approximately 3.8 degrees C) for the buffer used (10 mM Tris-HCI, pH 7.5) (beta is the thermal cubic volumetric expansion coefficient). For all of the proteins except for acrylodan-labeled IFABP, the fluorescence quantum yields calculated assuming simple energy conservation were anomalously high, i.e., the apparent heat signals were lower than those predicted from independent fluorescence measurements. The consistent anomalies suggest that the low photoacoustic signals may be characteristic of fluorophores buried in proteins, and that photoacoustic signals derive in part from the microenvironment of the absorbing chromophore.

2-Naphthylamine↗
PubMed1996

Affinity of fatty acid for (r)rat intestinal fatty acid binding protein:further examination.

The enhancement of the fluorescence quantum yield of 1,8-anilinonaphthalenesulfonic acid (ANS) upon binding to intestinal fatty acid protein (I-FABP) was exploited to devise an assay for free I-FABP. With this assay, we monitored the competition for free I-FABP between ANS and fatty acids and thereby extracted values for the dissociation constants (K(FA)) of fatty acids for I-FABP. We obtained these constants for the I-FABP ligands oleic acid, arachidonic acid, and palmitic acid. In addition, we measured the dependence of K(FA) for oleic acid upon temperature and at two pH values. From these data, we calculate the van't Hoff enthalpy of oleic acid binding. This enthalpy is compared with the enthalpies of binding obtained directly from titration calorimetry. Our experiments with the fluorescence-based assay generate values of K(FA) which disagree with older values obtained from calorimetry and other methods. Our own calorimetric data were analyzed with a view to improving the technique involved in subtraction of a "reference" dilution of the ligand into solution in the absence of the protein. By this maneuver, we obtained "corrected" titrations which could be fitted to values of K(FA) more in agreement with the values we determined via the fluorescence-based assay than wer the older literature values. Our new values for K(FA) also agree substantially with values derived using a complementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richiere et al [Richiere et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of delta H degrees, delta S degrees, and delta C(p)degrees for fatty acid binding we have obtained in this work with those we found in earlier work with ANS binding to I-FABP [Kirk et al. (1996) Biophys. J. 70, 69-83]. Our interpretation of the origin of the thermodynamic changes for ANS binding in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I-FABP with fatty acids.

Anilino Naphthalenesulfonates↗
PubMed1996

Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein.

1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity.

Anilino Naphthalenesulfonates↗
PubMed1996

Infrared reflection-absorption of melittin interaction with phospholipid monolayers at the air/water interface.

The interaction of melittin with monolayers of 1,2-dipalmitoylphosphatidylcholine and 1,2-dipalmitoylphosphatidylserine has been investigated with infrared external reflection-absorption spectroscopy. Improved instrumentation permits determination of acyl chain conformation and peptide secondary structure in situ at the air/water interface. The IR frequency of the 1,2-dipalmitoylphosphatidylcholine antisymmetric acyl chain CH2 stretching vibration decreases by 1.3 cm-1 upon melittin insertion, consistent with acyl chain ordering, whereas the same vibrational mode increases by 0.5 cm-1 upon peptide interaction with the 1,2-dipalmitoylphosphatidylserine monolayer, indicative of chain disordering. Thus the peptide interacts quite differently with zwitterionic compared with negatively charged monolayer surfaces. Melittin in the monolayer adopted a secondary structure with an amide l(l') frequency (1635 cm-1) dramatically different from the alpha-helical motif (amide l frequency 1656 cm-1 in a dry or H2O hydrated environment, amide l' frequency 1645 cm-1 in an H-->D exchanged alpha-helix) assumed in bilayer or multibilayer environments. This work represents the first direct in situ spectroscopic indication that peptide secondary structure in lipid monolayers may differ from that in bilayers.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Tryptophan dynamics of the FK506 binding protein: time-resolved fluorescence and simulations.

The FK506-binding protein (FKBP12) is important in the immunosuppressant action of FK506 and rapamycin. We have investigated Trp side chain dynamics in FKBP12, with and without a bound immunosuppressant, by measuring the Trp time-resolved fluorescence anisotropy decay r(t). The r(t) for W59 in aqueous uncomplexed FKBP12 at 20 degrees C is well described by a single exponential with a recovered initial anisotropy, r(eff)o, of 0.192 and an overall rotational correlation time for the protein, phi p, of 4.7 ns; r(eff)o = 0.214 and phi p = 4.2 ns for the FKBP12/FK506 complex. Using an expression for the order parameter squared, namely S2 = r(eff)o/rTo, where rTo is the vitrified steady-state excitation anisotropy, we recovered an S2 of 0.75 for W59 fluorescence in uncomplexed FKBP12 and S2 approximately equal to 1 in the FKBP12/FK506 complex. Results obtained for the FKBP12/rapamycin complex are similar to those found for the FKBP12/FK506 complex. Minimum perturbation mapping simulations were performed on the free and complexed forms of FKBP12 and the results were generally in agreement with the experimental data.

Biophysical Phenomena↗
PubMed1996

Structure and dynamics of melittin in lysomyristoyl phosphatidylcholine micelles determined by nuclear magnetic resonance.

Mixed micelles of the 26-residue, lytic peptide melittin (MLT) and 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) in aqueous solution at 25 degrees C were investigated by (13)C- and (31)P-NMR spectroscopy. (13)C alpha chemical shifts of isotopically labeled synthetic MLT revealed that MLT in the micelle is predominantly alpha-helical and that the peptide secondary structure is stable from pH 4 to pH 11. Although the helical transformation of MLT as determined from NMR is evident at lipid:peptide molar ratios as low as 1:2, tryptophan fluorescence measurements demonstrate that well-defined micellar complexes do not predominate until lipid:peptide ratios exceed 30:1. (31)P linewidth measurements indicate that the interaction between phosphate ions in solution and cationic groups on MLT is pH dependent, and that the phosphoryl group of MMPC senses a constant charge, most likely +2, on MLT from pH 4 to pH 10. (13)C-NMR relaxation data, analyzed using the model-free formalism, show that the peptide backbone of MLT is partially, but not completely, immobilized in the mixed micelles. Specifically, order parameters (S(2)) of C alpha-H vectors averaged 0.7 and were somewhat larger for residues in the N-terminal half of the molecule. The amino terminal glycine had essentially the same range of motion as the backbone carbons. Likewise, order parameters for the trp side chain were similar to those found for the peptide C alpha moieties, as was verified by trp fluorescence anisotropy decay data. In contrast, the motion of the lysine side chains was less restricted, the average S(2) values for the C epsilon-H vectors being 0.19, 0.30, and 0.44 for lys-7, 21, and 23, respectively, for MLT in the mixed micelles. Values of the effective correlation time of the local motion tau e were in the motional narrowing limit and usually longer for side-chain atoms than for those in the backbone. The dynamics were independent of pH from pH 4 to pH 9, but at pH 11 the correlation time for the rotational motion of the mixed micelles as a whole increased from 10 ns to 16 ns, and S(2) for the lys side chains increased. Overall it appears that the MLT helix lies near the surface of the micelle at low to neutral pH, but at higher pH its orientation changes, accompanied by deeper penetration of the lysine side chains into the micelle interior. It is apparent, however, that the MLT-lipid interaction is not dependent on deprotonation of any of the titratable cationic groups in the peptide in the pH 4-10 range, and that there is substantial backbone and side-chain mobility in micelle-bound MLT.

Amino Acid Sequence↗
PubMed1995

N-terminus and lysine side chain pKa values of melittin in aqueous solutions and micellar dispersions measured by 15N NMR.

Melittin (MLT) is a 26-amino acid cytolytic peptide from the Apis mellifera honey bee. It is known to exist as an alpha-helical tetramer, as an alpha-helical monomer, or as a monomeric random coil depending on solvent conditions. The charge state of MLT is believed to be a major factor in determining its aggregation properties and its interaction with lipids. Several, contradictory, indirect measurements of the pKa values of the three lysine groups in MLT have been reported. In the present study, high-resolution 15N NMR at 50.6 MHz was used to directly measure the pKa values of the amino groups of the Gly-1, Lys-7, Lys-21, and Lys-23 residues of MLT. Specifically, the pH dependence of MLT 15N chemical shifts was measured separately for the isotopically enriched backbone nitrogen of Gly-1 and the side chain nitrogen atoms of Lys-7, Lys-21, and Lys-23 at a MLT concentration of 1.2 mM and a temperature of 23 degrees C. Measurements were made for MLT in potassium phosphate buffer, in neat water, and in 1-myristoyl-2-hydroxyl-sn-glycero-3-phosphocholine (MMPC) lipid micelles. The experiments showed for MLT tetramer in aqueous phosphate buffer that the amino nitrogen of Gly-1 has a pKa of 8.15, and that the Lys-7, Lys-21, and Lys-23 side chain nitrogen atoms have pKa values of 10.21, 10.03, and 10.24 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Fluorescence of a tryptophan bearing peptide from smooth muscle myosin light chain kinase upon binding to two closely related calmodulins.

We have investigated the fluorescence of a calmodulin binding peptide (AS19) based on the sequence of the calmodulin binding domain of smooth muscle myosin light chain kinase and bearing a tryptophan residue at position 5 upon binding to two closely related calmodulins. The emission maximum of peptide AS19 bound to the engineered SYNCAM calmodulin was 318 nm and a vibrational structure was clearly apparent. The emission maximum of peptide AS19 bound to chicken gizzard calmodulin (ChG CaM) was 327 nm and its spectrum was featureless. Red edge excitation effect supports the assumption that the polarity of Trp-5 environment is larger in the complex with ChG CaM than with SYNCAM, in agreement with fluorescence spectra. Time-resolved fluorescence and anisotropy measurements showed that, in both complexes, the tryptophan emitting state was 1La. The X-ray structure of the calmodulin-peptide complex has been resolved (W. E. Meador, A. R. Means, and F. A. Quiocho, 1992, Science 257, 1251-1255). The Trp binding site has been characterized. It differs by a single-point mutation between the two calmodulins: Met-144 of ChG CaM has been replaced by Val in SYNCAM. This suggests that the spectral relaxation of Trp-5 in the complex with ChG CaM as compared to SYNCAM is due to the polarizability of the sulfur atom containing Met side chain that is higher than that of Val. This provides an ideal system to investigate the origin of the Stokes shift of the indole moiety in proteins.

Amino Acid Sequence↗
PubMed1995

Green-fluorescent protein mutants with altered fluorescence excitation spectra.

Using random mutagenesis and visual selection of fluorescent clones, we have isolated a T203I and a E222G mutant of the Aequorea green-fluorescent protein. Each mutant has one of the two fluorescence excitation bands of the wild type deleted and retains the other without a wavelength shift. This finding is consistent with each excitation band corresponding to a distinct spectroscopic state of the chromophore. Both mutations are single amino acid exchanges which in the linear sequence are located remotely from the chromophore but in the folded protein may be situated in its vicinity. We conclude that the mutations influence the fluorescence properties by changing the interactions between the chromophore and its protein environment.

Amino Acid Sequence↗
PubMed1995

Analytical approach to the recovery of short fluorescence lifetimes from fluorescence decay curves.

Considerable effort in instrument development has made possible detection of picosecond fluorescence lifetimes by time-correlated single-photon counting. In particular, efforts have been made to narrow markedly the instrument response function (IRF). Less attention has been paid to analytical methods, especially to problem of discretization of the convolution integral, on which the detection and quantification of short lifetimes critically depends. We show that better discretization methods can yield acceptable results for short lifetimes even with an IRF several times wider than necessary for the standard discretization based on linear approximation (LA). A general approach to discretization, also suitable for nonexponential models, is developed. The zero-time shift is explicitly included. Using simulations, we compared LA, quadratic, and cubic approximations. The latter two proved much better for detection of short lifetimes and, in that respect, they do not differ except when the zero-time shift exceeds two channels, when one can benefit from using the cubic approximation. We showed that for LA in some cases narrowing the IRF beyond FWHM = 150 ps is actually counterproductive. This is not so for quadratic and cubic approximations, which we recommend for general use.

Kinetics↗
PubMed1994

Coupling between external viscosity and the intramolecular dynamics of ribonuclease T1: a two-phase model for the quenching of protein fluorescence.

Our recent equilibrium dialysis studies showed that proteins are able to interact preferentially with acrylamide (Punyiczki et al. (1993) Biophys. Chem. 47, 9-19). The presence of considerable amounts of acrylamide--albeit weakly bound--in the protein volume, coupled with the failure of a simple gating model of quenching to rationalise viscosity dependence of the quenching of tryptophan (Trp) fluorescence in Ribonuclease T1 (RNase T1) has prompted us to explore a new model, the two-phase model for quenching. According to this model, the dynamic quenching is accomplished by quencher molecules already in the protein phase at the moment of excitation. Some of the molecules may, at this moment, form an encounter complex with the fluorophore and thus be responsible for the observed static contribution. We use the rate equation derived from our model to study the viscosity dependence of acrylamide quenching of Trp fluorescence in RNase T1. The model allows us to separate co-solvent effects: the chemical effect on the protein and on the distribution of quencher molecules between the bulk and the protein phases and, further, the viscosity effect due to coupling between the bulk viscosity and the local friction affecting intramolecular fluctuations of the protein matrix. We express local friction in terms of bulk viscosity, eta, and a coupling constant kappa (friction = eta kappa). Addition of glycerol up to 65% is characterised by a kappa of 0.50. The viscosity dependence of the apparent bimolecular quenching constant is a combination of two compensating effects: changes in chemical activity and changes in patterns of structural fluctuations.

Acrylamide↗
PubMed1994

Mechanism of aldose reductase inhibition: binding of NADP+/NADPH and alrestatin-like inhibitors.

Aldose reductase enfolds NADP+/NADPH via a complex loop mechanism, with cofactor exchange being the rate-limiting step for the overall reaction. This study measures the binding constants of these cofactors in the wild-type enzyme, as well as a variety of active-site mutants (C298A, Y48H, Y48F, Y209F, H110A, W219A, and W20A), and seeks to identify the binding site and mechanism of the aldose reductase inhibitor alrestatin in the recombinant human enzyme. All the mutant enzymes, regardless of their enzyme activities, have normal or only slightly elevated coenzyme binding constants, suggesting a tertiary structure similar to that of the wild-type enzyme. Binding of alrestatin was detected by fluorescence assays, and by an ultrafiltration assay which measures the fraction of unbound alrestatin. Alrestatin binds preferentially to the enzyme/NADP+ complex, consistent with the steady-state inhibition pattern. Alrestatin binding and enzyme inhibition were abolished in the Tyr48 mutants Y48F and Y48H, implicating the positively charged anion well formed by the Asp43-/Lys77+/Tyr48(0)/NADP+ complex in inhibitor binding (Harrison et al., 1994; Bohren et al., 1994). The enzyme mutant W20A severely affected the inhibitory potencies of a variety of commercially developed aldose reductase inhibitors (zopolrestat, tolrestat, FK366, AL1576, alrestatin, ponalrestat, and sorbinil). Inhibition by citrate, previously shown to bind to the positively charged anion well, was not affected by this mutation. Inhibitors with flexible double aromatic ring systems (Zopolrestat, FK366, and ponalrestat) were less affected than others possessing a single aromatic ring system, suggesting that the additional pharmacophor ring system stabilizes the inhibitor by interaction at some other hydrophobic site.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Reductase↗
PubMed1994

Calmodulin interacts with amphiphilic peptides composed of all D-amino acids.

Calmodulin binds to amphiphilic, helical peptides of a variety of amino-acid sequences. These peptides are usually positively charged, although there is spectroscopic evidence that at least one neutral peptide binds. The complex between calmodulin and one of its natural target peptides, the binding site for calmodulin on smooth muscle myosin light-chain kinase (RS20), has been investigated by crystallography and NMR which have characterized the interactions between the ligand and the protein. From these data, it appears that the calmodulin-binding surface is sterically malleable and van der Waals forces probably dominate the binding. To explore further this apparently permissive binding, we investigated the chiral selectivity of calmodulin using synthesized analogues of melittin and RS20 that consisted of only D-amino acids. Fluorescence and NMR measurements show that D-melittin and D-RS20 both bind avidly to calmodulin, probably in the same general binding site as that for peptides having all L-amino acids. The calmodulin-peptide binding surface is therefore remarkably tolerant sterically. Our results suggest a potentially useful approach to the design of non-hydrolysable or slowly hydrolysable intracellular inhibitors of calmodulin.

Binding Sites↗