Structure of the influenza C virus CM2 protein transmembrane domain obtained by site-specific infrared dichroism and global molecular dynamics searching

The 115-residue protein CM2 from Influenza C virus has been recently characterized as a tetrameric integral membrane glycoprotein. Infrared spectroscopy and site-directed infrared dichroism were utilized here to determine its transmembrane structure. The transmembrane domain of CM2 is alpha-helical, and the helices are tilted by beta = (14.6 +/- 3.0) degrees from the membrane normal. The rotational pitch angle about the helix axis omega for the 1-(13)C-labeled residues Gly(59) and Leu(66) is omega = (218 +/- 17) degrees, where omega is defined as zero for a residue pointing in the direction of the helix tilt. A detailed structure was obtained from a global molecular dynamics search utilizing the orientational data as an energy refinement term. The structure consists of a left-handed coiled-coil with a helix crossing angle of Omega = 16 degrees. The putative transmembrane pore is occluded by the residue Met(65). In addition hydrogen/deuterium exchange experiments show that the core is not accessible to water.     

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1,N6-ethenoadenylic acid)

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1,N6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1,N6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(+/- 6)degrees. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 degrees with the local helix axis. Some possible structures of the DNA-protein complex are discussed.     

vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching.

The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain.     

Dramatic in situ conformational dynamics of the transmembrane protein bacteriorhodopsin.

The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method.     

Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel

The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure.     

Structural basis for SRY-dependent 46-X,Y sex reversal: modulation of DNA bending by a naturally occurring point mutation

The HMG-box domain of the human male sex-determining factor SRY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequence-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRY(HMG) construct, acts principally by reducing the extent of DNA bending. To elucidate the structural consequences of the M9I mutation, we have solved the 3D solution structures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, including the use of residual dipolar couplings to derive long-range orientational information. We show that the average bend angle (derived from an ensemble of 400 simulated annealing structures for each complex) is reduced by approximately 13 degrees from 54(+/-2) degrees in the wild-type complex to 41(+/-2) degrees in the M9I complex. The difference in DNA bending can be localized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13. The larger bend angle in the wild-type complex arises as a direct consequence of steric repulsion of the sugar of the second adenine by the bulky S(delta) atom of Met9, whose position is fixed by a hydrogen bond with the guanidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar moieties of the two adenine residues, thereby decreasing the roll and tilt angles at the ApA step by approximately 8 degrees and approximately 5 degrees, respectively, and resulting in an overall difference in bend angle of approximately 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protein-DNA complex have been visualized at the atomic level.     

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.     

Structure and dynamics of membrane-associated ICP47, a viral inhibitor of the MHC I antigen-processing machinery

To evade the host's immune response, herpes simplex virus employs the immediate early gene product ICP47 (IE12) to suppress antigen presentation to cytotoxic T-lymphocytes by inhibition of the ATP-binding cassette transporter associated with antigen processing (TAP). ICP47 is a membrane-associated protein adopting an alpha-helical conformation. Its active domain was mapped to residues 3-34 and shown to encode all functional properties of the full-length protein. The active domain of ICP47 was reconstituted into oriented phospholipid bilayers and studied by proton-decoupled 15N and 2H solid-state NMR spectroscopy. In phospholipid bilayers, the protein adopts a helix-loop-helix structure, where the average tilt angle of the helices relative to the membrane surface is approximately 15 degrees (+/- 7 degrees ). The alignment of both structured domains exhibits a mosaic spread of approximately 10 degrees . A flexible dynamic loop encompassing residues 17 and 18 separates the two helices. Refinement of the experimental data indicates that helix 1 inserts more deeply into the membrane. These novel insights into the structure of ICP47 represent an important step toward a molecular understanding of the immune evasion mechanism of herpes simplex virus and are instrumental for the design of new therapeutics.     

Orientation of the bases of single-stranded DNA and polynucleotides in complexes formed with the gene 32 protein of bacteriophage T4. A linear dichroism study

Linear dichroism measurements were performed in the wavelength region 250 to 350 nm on complexes between the single-stranded DNA binding protein of bacteriophage T4 (gp32) and single-stranded DNA and a variety of homopolynucleotides in compressed polyacrylamide gels. The complexes appeared to orient well, giving rise to linear dichroism spectra that showed contributions from both the protein aromatic residues and the bases of the polynucleotides. In most cases the protein contribution appeared to be very similar, and the linear dichroism of the bases could be explained by similar orientations of the bases for most of the complexes. Assuming a similar, regular structure for most of the polynucleotides in complex, only a limited set of combinations of tilt and twist angles can explain the linear dichroism spectra. These values of tilt and twist are close to (-40 degrees, 30 degrees), (-40 degrees, 150 degrees), (40 degrees, -30 degrees) or (40 degrees, -150 degrees), with an uncertainty in both angles of about 15 degrees. Although the linear dichroism results do not allow a choice between these possible orientations, the latter two combinations are not in agreement with earlier circular dichroism calculations. For the complexes formed with poly(rC) and poly(rA), the linear dichroism spectra could not be explained by the same base orientations. In these two cases also the protein contribution to the linear dichroism appeared to be different, indicating that for some aromatic residues the orientations are not the same as those in the other complexes. The different structures of these complexes are possibly related to the relatively low binding affinity of gp32 to poly(rC), and to a lesser extent to poly(rA).     

Structural and orientational information of the membrane embedded M13 coat protein by (13)C-MAS NMR spectroscopy

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by (13)C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Calpha and C&z.dbnd6;O positions of the labelled residues Val-29/Val-30 (0.4+/-0.5nm) and Val-29/Val-31 (0.45+/-0. 5nm) in its hydrophobic domain. The derived dihedral angles (Phi, Psi) for Val-30 revealed a local alpha-helical conformation. (13)C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the (13)C&z.dbnd6;O labelled site of Val-30 allowed the determination of the helix tilt (20 degrees +/-10 degrees ) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.     

The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.     

Macromolecular organization of collagen fibres in natural and tanned basement membrane

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.     

Structure, dynamics, and membrane topology of stannin: a mediator of neuronal cell apoptosis induced by trimethyltin chloride

Organotin compounds or alkyltins are ubiquitous environmental toxins that have been implicated in cellular death. Unlike other xenobiotic compounds, such as organomercurials and organoleads, alkyltins activate apoptotic cascades at low concentrations. Trimethyltin (TMT) chloride is amongst the most toxic organotin compounds, and is known to selectively inflict injury to specific regions of the brain. Stannin (SNN), an 88-residue mitochondrial membrane protein, has been identified as the specific marker for neuronal cell apoptosis induced by TMT intoxication. This high specificity of TMT makes SNN an ideal model system for understanding the mechanism of organotin neurotoxicity at a molecular level. Here, we report the three-dimensional structure and dynamics of SNN in detergent micelles, and its topological orientation in lipid bilayers as determined by solution and solid-state NMR spectroscopy. We found that SNN is a monotopic membrane protein composed of three domains: a single transmembrane helix (residues 10-33) that transverses the lipid bilayer at approximately a 20 degrees angle with respect to the membrane normal; a 28 residue unstructured linker, which includes a conserved CXC metal-binding motif and a putative 14-3-3zeta binding domain; and a distorted cytoplasmic helix (residues 61-79) that is partially absorbed into the plane of the lipid bilayer with a tilt angle of approximately 80 degrees from the membrane normal. The structure and architecture of SNN within the lipid environment provides insight about how this protein transmits toxic insults caused by TMT across the membrane.     

A structure for the trimeric MHC class II-associated invariant chain transmembrane domain

The major histocompatibility complex (MHC)-associated invariant chain (Ii) contains a single transmembrane domain that forms trimers. Ii is involved in the assembly of the MHC and antigen presentation, and is thus central to the function of the immune system. Here, we show by attenuated total reflectance, Fourier transform infrared (ATR-FTIR) spectroscopy that the transmembrane domain is alpha-helical and we provide a structural model of the transmembrane domain obtained by a combination of site-specific infrared dichroism and molecular dynamics (MD) simulations. This work resolves the backbone structure of a transmembrane peptide by multiple (13)C=(18)O labelling at ten different residues. A second purely computational approach, based on MD simulations of Ii transmembrane homologous sequences, yields a similar structure that is consistent with our experimental results. The structure presented forms a left-handed coiled coil with an average helix tilt of 13(+/-6) degrees; the residue Gln47 implicated in trimer formation forms strong interhelical contacts, Thr50 points to the inside of the trimeric coil and forms a network of hydrogen bonds.     

Use of a single glycine residue to determine the tilt and orientation of a transmembrane helix. A new structural label for infrared spectroscopy

Site-directed dichroism is an emerging technique for the determination of membrane protein structure. However, due to a number of factors, among which is the high natural abundance of (13)C, the use of this technique has been restricted to the study of small peptides. We have overcome these problems through the use of a double C-deuterated glycine as a label. The modification of a single residue (Gly) in the transmembrane segment of M2, a protein from the Influenza A virus that forms H(+)-selective ion channels, has allowed us to determine its helix tilt and rotational orientation. Double C-deuteration shifts the antisymmetric and symmetric stretching vibrations of the CD(2) group in glycine to a transparent region of the infrared spectrum where the dichroic ratio of these bands can be measured. The two dichroisms, along with the helix amide I dichroic ratio, have been used to determine the helix tilt and rotational orientation of M2. The results are entirely consistent with previous site-directed dichroism and solid-state NMR experiments, validating C-deuterated glycine (GlyCD(2)) as a structural probe that can now be used in the study of polytopic membrane proteins.     

Conformation of alamethicin in oriented phospholipid bilayers determined by (15)N solid-state nuclear magnetic resonance

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.     

In meso structure of the cobalamin transporter, BtuB, at 1.95 A resolution

Crystals of the apo form of the vitamin B12 and colicin receptor, BtuB, that diffract to 1.95 A have been grown by the membrane-based in meso technique. The structure of the protein differs in several details from that of its counterpart grown by the more traditional, detergent-based (in surfo) method. Some of these differences include (i) the five N-terminal residues are resolved in meso, (ii) residues 57-62 in the hatch domain and residues 574-581 in loop 21-22 are disordered in meso and are ordered in surfo, (iii) residues 278-287 in loop 7-8 are resolved in meso, (iv) residues 324-331 in loop 9-10, 396-411 in loop 13-14, 442-458 in loop 15-16 and 526-541 in loop 19-20 have large differences in position between the two crystal forms, as have residues 86-96 in the hatch domain, and (v) the conformation of residues 6 and 7 in the Ton box (considered critical to signal transduction and substrate transport) are entirely different in the two structures. Importantly, the in meso orientation of residues 6 and 7 is similar to that of the vitamin B12-charged state. These data suggest that the "substrate-induced" 180 degrees -rotation of residues 6 and 7 reported in the literature may not be a unique signalling event. The extent to which these findings agree with structural, dynamic and functional insights gleaned from site-directed spin labelling and electron paramagnetic resonance measurements is evaluated. Packing in in meso grown crystals is dense and layered, consistent with the current model for crystallogenesis of membrane proteins in lipidic mesophases. Layered packing has been used to locate the transmembrane hydrophobic surface of the protein. Generally, this is consistent with tryptophan, tyrosine, lipid and CalphaB-factor distributions in the protein, and with predictions based on transfer free energy calculations.     

Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1

The three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu(2-30+) is a 36-residue polypeptide that consists of residues 2-30 from the N terminus of Vpu and a six-residue "solubility tag" at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/15N HSQC spectrum of uniformly 15N labeled Vpu(2-30+) in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/15N PISEMA spectrum of Vpu(2-30+) in lipid bilayers aligned between glass plates has been fully resolved and assigned. The "wheel-like" pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu(2-30+) has a trans-membrane alpha-helix spanning residues 8-25 with an average tilt of 13 degrees. The helix is kinked slightly at Ile17, which results in tilts of 12 degrees for residues 8-16 and 15 degrees for residues 17-25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 A. Vpu(2-30+) exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(+/- 6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.     

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.     

Helix tilt of the M2 transmembrane peptide from influenza A virus: an intrinsic property

Solid-state NMR has been used to study the influence of lipid bilayer hydrophobic thickness on the tilt of a peptide (M2-TMP) representing the transmembrane portion of the M2 protein from influenza A. Using anisotropic (15)N chemical shifts as orientational constraints, single-site isotopically labeled M2-TMPs were studied in hydrated dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine (DMPC) lipid bilayers oriented between thin glass plates. These chemical shifts provide orientational information for the molecular frame with respect to the magnetic field in the laboratory frame. When modeled as a uniform ideal alpha-helix, M2-TMP has a tilt of 37(+/-3) degrees in DMPC and 33(+/-3) degrees in DOPC with respect to the bilayer normal in these lipid environments. The difference in helix tilt between the two environments appears to be small. This lack of a substantial change in tilt further suggests that significant interactions occur between the helices, as in an oligomeric state, to prevent a change in tilt in thicker lipid bilayers.