No abstract

The secondary structure, amide hydrogen exchangeability, and intramembrane orientation of the hydrophobic peptide Ac-K2-(LA)12-K2-amide [(LA)12] were studied by a combination of circular dichroism (CD), Fourier transform infrared (FTIR), and proton nuclear magnetic resonance (1H NMR) spectroscopic techniques. All three techniques indicate that (LA)12 adopts predominantly helical conformations in various organic solvents, detergent micelles, and phospholipid bilayers. Also, attenuated total reflectance FTIR studies of oriented phospholipid bilayers demonstrate that (LA)12 is arranged with the long helical axis perpendicular to the bilayer plane. FTIR and 1H NMR studies of the exchangeability of the amide protons of (LA)12 indicate that in all media there are at least two populations of amide protons which exchange with the bulk solvent at markedly different rates. Moreover, the 1H NMR spectroscopic studies indicate that, in organic solvents and micellar dispersions, amide proton exchange rates decrease progressively from the N- or C-terminus of the peptide toward the central region. Our results are thus consistent with (LA)12 retaining a predominantly helical structure with so-called frayed ends in all media. The amide proton exchange studies also indicate that when (LA)12 is dispersed in lipid bilayers, the slowly exchanging population of amide protons is larger than that observed in organic solvents or in micellar dispersions and that most of that proton population is virtually unexchangeable. Such observations are consistent with the sequestration of the central regions of the peptide in the hydrophobic domains of the lipid bilayer. The CD and FTIR data indicate that although (LA)12 seems to retain conformations with a high alpha-helical content in all media examined, its conformation is sensitive to the composition of the surrounding medium, in contrast to the polyleucine-based analogues which have been studied previously. In particular, the FTIR spectroscopic data indicate that (LA)12 may exhibit an amide I absorption band between 1633 and 1639 cm-1 under some circumstances. The relative intensity of this band changes with the composition of the surrounding medium and its appearance has previously been correlated with the formation of 3(10)-helical structures [Miick et al. (1992) Nature 359, 653-655]. Thus (LA)12 may be interconverting between different helical conformations in response to changes in the physical properties of the medium in which the peptide is dispersed. Our results suggest that (LA)12 should serve as a good peptide model of hydrophobic, transmembrane helices which are conformationally sensitive to the properties of the lipid bilayer in which they reside.

The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection. 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles. A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of [3-13C]alanine. Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total). Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different. The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems. The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure. Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e. The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution proton N M R spectrocopy has been used to study the solution structures of the subfragment 1 (SI) isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra are unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted in solution. These observations are in agreement with previous studies of the mixed isoenzymes [Highsmith et al. (1 979) Biochemistry, 18, 4238 -42431. Peptide amide exchange studies show also that the S 1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale-of the 'H-NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility as well as domains of different sidechain mobility.Close comparison of the Sl(A1) and Sl(A2) spectra indicate that the N-terminal41 residues of the A1 light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [SI(AI)-Sl(A2)] obtained closely resembles the spectral simulation of the 41-residue segment. Upon addition of actin, many of the narrow S1 resonances decrease in intensity or progressively disappear altogether, indicative of intermediateslow exchange conditions consistent with the recognised high affinity between the two proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1 as has been suggested in earlier H-NMR studies referred to above. In particular, the differences noted between S l(A 1 ) and S l(A2) have now largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N-terminal region of the light chain in S l(A 1). Together with other affinity chromatography results [Winstanley and Trayer (1 979) Biochem. Soc. Trans. 7, 703 -7041, this is good evidence for a direct interaction between this area of S l(A 1) and actin.The mechanism of muscle contraction, as originally proposed by Huxley [I] and modified by several groups of authors [2, 31, consists of a cycle of cross-bridge detachments and attachments, which are the basis of the 'sliding filament' theory, The myosin heads form cross-bridges with actin, which are then detached by ATP. The hydrolysis of ATP produces some conformational change in myosin which is restored as mechanical energy when the heads rebind actin. There is now ample evidence that this theory is correct. There are, however, considerable differences of opinion with regard to the intermolecular and intramolecular interactions involved in the actomyosin complex, and several models have been proposed, as reviewed by Taylor [4]. X-ray diffraction and electron microscopy indicate that the cross-bridge or part of it rotates during contraction [5] and the existence of a 'swivel' and a 'hinge' have been postulated in myosin [6 -1 I...

The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.

The dynamic behaviour of the guanidino group of arginine has been investigated quantitatively with the intention of providing a set of basis values for the interpretation of data acquired for arginine residues in proteins. At room temperature, a single broad resonance line is observed for the four η-NH(2) protons. Upon cooling the sample (≈10°C at 500 MHz), two η-NH(2) proton resonances are resolved which were shown by HMQC spectroscopy to be the result of slowed rotation about the N(ε)-C(ζ) partial double bond. The flip rate (k(NC)) about the N(ε)-C(ζ) bond was measured as a function of temperature using line-shape analysis of both (1)H and (15)N NMR spectra; at 25°C, k(NC) is between 900 and 1000 s(-1). The exchange broadening, due to N(ε)-C(ζ) bond flips, typically results in weak or missing signals for the η-NH(2) protons of arginine residues in HMQC or INEPT experiments recorded at room temperature, unless the motion is restricted in some way. In a related series of experiments, the pH dependence of the hydrogen exchange rates of the ε-NH and η-NH(2) protons of arginine was measured using saturation transfer (1)H NMR spectroscopy and compared with the equivalent NH(2) protons of the guanidinium ion. As expected, OH ion catalysis dominates over most of the pH range and proceeds at a rate close to the diffusion limit for both types of proton (k(OH)=2×10(9)-1×10(10) M(-1)s(-1), depending on conditions). At low pH values, however, catalysis by H(3)O(+) becomes important and leads to characteristic rate minima in the exchange versus pH profiles. Acid catalysis is significantly more effective for the η-NH(2) protons than for the ε-NH proton; at low ionic strength (50 mM KCl) the rate minima occurred at pH 3.6 and 2.3, respectively. Under these conditions, acid-catalysed rate constants (k(H)) of 706 M(-1)s(-1) (η-NH(2)) and 3 M(-1)s(-1) (ε-NH) were obtained at 25°C. At high ionic strength (1 M KCl) the rate of OH(-) ion catalysis is decreased slightly, whereas the H(3)O(+)-catalysed rate is unchanged. The k(OH) value of the free guanidinium ion is identical to that of the η-NH(2) protons but acid catalysis occurs less easily, leading to a rate minimum at pH 3.3.

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

The E6 protein from high-risk types of human papillomavirus (HPV) binds PDZ-domain containing proteins and targets them for degradation. We used isothermal titration calorimetry to measure the interaction of a peptide from the C-terminus of HPV-18 E6 to the second PDZ domain (PDZ2) from the human homologue of the Drosophila discs large tumor suppressor protein (hDlg). Isothermal titration calorimetry experiments with a series of peptides showed that HPV-18 E6 bound hDlg PDZ2 about 5-fold stronger than HPV-16 E6, that the contribution of Arg154 to binding was about 1 kcal/mol, and that the binding was disabled by phosphorylation at Thr156. We then used NMR to determine the solution structure of the complex of PDZ2 bound to the HPV-18 E6 peptide. The resultant structures were of high quality and had backbone root-mean-square deviations of less than 0.5 A. The structure shows a novel mode of interaction in which six residues of the HPV-18 E6 peptide are contacted by the PDZ2 domain, in contrast to the typical four residues used by class I PDZ domains. Molecular dynamics simulations supported a model in which the C- and N-terminal ends of the peptide had different mobilities within the complex. Comparison of the NMR complex structure to previously determined X-ray structures of PDZ2 by itself and bound to different peptides allows a description of conformational changes required for PDZ2 to bind to HPV-18 E6.