Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.     

Structural roles of subunit cysteines in the folding and assembly of the DNA packaging machine (portal) of bacteriophage P22

The DNA packaging machine (portal assembly) of bacteriophage P22 is constructed from 12 copies of a multidomain 725-residue subunit comprising a complex alpha/beta fold. The portal subunit contains four cysteines (Cys 153, Cys 173, Cys 283, and Cys 516), which produce distinctive Raman markers in the spectral interval 2500-2600 cm(-1) originating from S-H bond-stretching vibrations diagnostic of S-H...X hydrogen-bonding interactions. The Raman spectrum is unique in the capability to characterize cysteine sulfhydryl interactions in proteins and shows that portal cysteine environments are significantly altered by assembly (Rodriguez-Casado et al. (2001) Biochemistry 40, 13583-13591). We have employed site-directed mutagenesis, size-exclusion chromatography, and Raman difference spectroscopy to characterize the roles of portal cysteines in subunit folding and dodecamer assembly. The stability of the portal monomer is severely reduced by a Cys --> Ser point mutation introduced at either residue 173 or 516. In the case of C516S, the destabilized monomer still forms portal rings, as visualized by negative-stain electron microscopy, whereas portal ring formation cannot be detected for C173S, which forms aberrant aggregates. The C283S mutant is a hyperstable monomer that is defective in portal ring formation. Interestingly, Cys 283 is suggested by secondary structure homology with the phi29 portal to be within a domain involved in DNA translocation. Conversely, the phenotype of the C153S mutant is close to that of the wild-type protein, implying that the sulfhydryl moiety of Cys 153 is not essential to formation of the native subunit fold and productive assembly dynamics. The present results demonstrate that cysteines of the P22 portal protein span a wide range of sulfhydryl hydrogen-bonding strengths in the wild-type assembly, that three of the four sulfhydryls play key roles in portal protein stability and assembly kinetics, and that substitution of a mutant seryl interaction (O-H...X) for a wild-type cysteinyl interaction (S-H...X) can either stabilize or destabilize the native fold depending upon sequence context.     

Assembly dynamics of the nucleocapsid shell subunit (P8) of bacteriophage phi6.

Phi6 is an enveloped dsRNA bacteriophage of Pseudomonas syringae. The viral envelope encloses a nucleocapsid, consisting of an RNA-dependent RNA polymerase complex within an icosahedral shell assembled from approximately 800 copies of a 16 kDa subunit (protein P8, encoded by viral gene 8). During infection, the nucleocapsid penetrates the host plasma membrane and enters the cytosol, whereupon the P8 shell disassembles and the polymerase complex is activated. To understand the molecular mechanisms of shell assembly and disassembly-processes that have counterparts in most viral infections-we have investigated the structure, stability, and dynamics of P8 in different assembly states using time-resolved Raman spectroscopy and hydrogen-isotope exchange. In the presence of Ca(2+), which promotes shell assembly, the highly alpha-helical conformation of the P8 subunit is stabilized by rapid assembly into shell-like structures. However, in the absence of Ca(2+), the P8 subunit is thermolabile and unstable, manifested by a slow alpha-helix --> beta-strand conformational change and the accumulation of aberrant aggregates. In both properly assembled shells and aberrant aggregates, the P8 subunit retains an alpha-helical core that is protected against deuterium exchange of amide NH groups. Surprisingly, no additional protection against amide exchange is conferred by the shell lattice. Time-resolved assembly and disassembly experiments in deuterated buffers indicate that the regions of P8 involved in subunit/subunit interactions in the intact shell undergo rapid exchanges, presumably due to local unfolding events that are characterized by low activation barriers. Such localized dynamics of P8 within the shell lattice may mediate the nucleocapsid/host membrane interactions that are required in the cytosol for particle assembly during maturation and disassembly during infection.     

Domain structures and roles in bacteriophage HK97 capsid assembly and maturation

Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.     

Raman markers of nonaromatic side chains in an alpha-helix assembly: Ala, Asp, Glu, Gly, Ile, Leu, Lys, Ser, and Val residues of phage fd subunits

The study of filamentous virus structure by Raman spectroscopy requires accurate band assignments. In previous work, site- and residue-specific isotope substitutions were implemented to elucidate definitive assignments for Raman bands arising from vibrational modes of the alpha-helical coat protein main chain and aromatic side chains in the class I filamentous phage, fd [Overman, S. A., and Thomas, G. J., Jr. (1995) Biochemistry 34, 5440-5451; Overman, S. A., and Thomas, G. J., Jr. (1998) Biochemistry 37, 5654-5665]. Here, we extend the previous methods and expand the assignment scheme to identify Raman markers of nonaromatic side chains of the coat protein in the native fd assembly. This has been accomplished by Raman analysis of 11 different fd isotopomers selectively incorporating deuterium at specific sites in either alanine, aspartic acid, glutamic acid, glycine, isoleucine, leucine, lysine, serine, or valine residues of the coat protein. Raman markers are also identified for the corresponding deuterated side chains. In combination with previous assignments, the results provide a comprehensive understanding of coat protein contributions to the Raman signature of the fd virion and validate Raman markers assigned to the packaged single-stranded DNA genome. The findings described here show that nonaromatic side chains contribute prolifically to the fd Raman signature, that marker bands for specific nonaromatics differ in general from those observed in corresponding polypeptides and amino acids, and that the frequencies and intensities of many nonaromatic markers are sensitive to secondary and higher-order structures. Nonaromatic markers within the 1200-1400 cm-1 interval also interfere seriously with the diagnostic Raman amide III band that is normally exploited in secondary structure analysis. Implications of these findings for the assessment of protein conformation by Raman spectroscopy are considered.     

New structural insights from Raman spectroscopy of proteins and their assemblies

Protein structure and stability are sensitive to and dependent on the local interactions of amino acid side chains. A diverse and important type of side-chain interaction is the hydrogen bond. Although numerous hydrogen bonds are resolved in protein 3-dimensional structures, those of the cysteine sulfhydryl group (S-H) are elusive to high-resolution X-ray and NMR methods. However, the nature and strength of sulfhydryl hydrogen bonds (S-H* * *X) are amenable to investigation by Raman spectroscopy. The power of the Raman method for characterizing S-H* * *X interactions is illustrated by resolving the Raman S-H stretching band for each of the eight cysteines per 666-residue subunit in the trimeric tailspike of icosahedral bacteriophage P22. The Raman sulfhydryl signatures of the wild-type tailspike and eight single-site cysteine to serine mutants reveal a heretofore unrecognized diversity of S-H hydrogen bonds in a native protein. The use of Raman spectroscopy to identify the non-hydrogen-bonded state of the tyrosine phenoxyl group is also described. This unusual and unexpected state occurs for all tyrosines in the assembled capsids of filamentous viruses Ff and Pf1. The Raman spectral signature of the non-hydrogen-bonded tyrosine phenoxyl, which is characterized by an extraordinary Raman Fermi doublet intensity ratio (I850/I830 = 6.7), extends and refines the existing correlation for hydrogen-bonded tyrosines. Finally, a novel Raman signature for tryptophan in the Pf3 filamentous virus is identified, which is proposed as diagnostic of "cation-pi interaction" involving the guanidinium group of Arg 37 as a cation donor and the indolyl ring of Trp 38 as a pi-electron acceptor. These studies demonstrate the power of Raman spectroscopy for investigating the interactions of key side chains in native protein assemblies.     

Structure and dynamics of the DNA-binding protein HU of B. stearothermophilus investigated by Raman and ultraviolet-resonance Raman spectroscopy

The histone-like protein HU of Bacillus stearothermophilus (HUBst) is a 90-residue homodimer that binds nonspecifically to B DNA. Although the structure of the HUBst:DNA complex is not known, the proposed DNA-binding surface consists of extended arms that project from an alpha-helical platform. Here, we report Raman and ultraviolet-resonance Raman (UVRR) spectra diagnostic of subunit secondary structures and indicative of key side-chains lining the proposed DNA-binding surface. Raman conformation markers show that the DNA-binding arms of the dimer contain beta-stranded structure in excess (eight +/- two residues per subunit) of that reported previously. Important among side-chain markers are Met (701 cm(-1)), Ala (908 cm(-1)), Arg (1082 cm(-1)), and Pro (1457 cm(-1)). The Ala marker undergoes a substantial shift (908 --> 893 cm(-1)) on deuteration of alanyl peptide sites, indicating a coupled side-chain/main-chain mode of diagnostic value in the identification of exchange-protected alanines. A large subset of alanines (67%) in the alpha-helical core exhibits robust resistance to exchange. A quantitative study of NH --> ND exchange exploiting newly identified amide II' markers of helical (1440 cm(-1)) and nonhelical (1472 cm(-1)) conformations of HUBst indicates unexpected flexibility at the dimer interface, which is manifested in rapid exchange of 80% of peptide sites. The results establish a basis for subsequent Raman and UVRR investigations of HUBst:DNA complexes and provide a framework for applications to other DNA-binding architectural proteins.     

Structure and organization of bacteriophage Pf3 probed by Raman and ultraviolet resonance Raman spectroscopy

The Pseudomonas bacteriophage Pf3 is a long and narrow filament consisting of a covalently closed DNA single strand of 5833 bases sheathed by approximately 2500 copies of a 44-residue subunit. Ultraviolet resonance Raman spectra excited at 257, 244, 238, and 229 nm and off-resonance Raman spectra excited at 514.5 nm are reported for Pf3 in both H2O and D2O solutions. The key Raman bands are assigned to specific protein and DNA groups of the native virion assembly. The results are compared with proposed assembly models and Raman spectra recently reported for the isomorphous (class II) Pseudomonas phage Pf1 and the morphologically distinct (class I) coliphage fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J. , Jr. (1997) Biochemistry 36, 7810-7820; Wen, Z. Q., Armstrong, A., and Thomas, G. J., Jr. (1999) Biochemistry 38, 3148-3156]. Surprisingly, deoxynucleosides of the packaged DNA genome of Pf3 adopt the same conformation (C3'-endo/anti) found for DNA packaged in the class I fd virion rather than that (C2'-endo/anti) associated with DNA in the isomorphous Pf1 virion. However, DNA base stacking in Pf3, as judged by Raman hypochromic effects, differs significantly from that occurring in either Pf1 or fd. Thus, the single-stranded DNA genomes of Pf3, Pf1, and fd are all organized differently within their respective capsids, implying that local subunit-DNA interactions may be important in determining the structure specific to each native assembly. The present study confirms a completely alpha-helical secondary structure for the Pf3 subunit and an unusual indolyl ring environment for the subunit tryptophan residue (Trp-38).     

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.     

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.     

Small terminase couples viral DNA binding to genome-packaging ATPase activity

Packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large (L) and a small (S) terminase subunit complexed with a dodecamer of portal protein. Here we describe the 1.75?? crystal structure of the bacteriophage P22 S-terminase in a nonameric conformation. The structure presents a central channel ～23?? in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of S-terminase are essential for binding to DNA and assembly to L-terminase. Upon binding to its own DNA, S-terminase functions as a specific activator of L-terminase ATPase activity. The DNA-dependent stimulation of ATPase activity thus rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits?a model for DNA-dependent activation of genome-packaging motors of general interest in virology.     

Ultraviolet-resonance raman spectroscopy of the filamentous virus Pf3: interactions of Trp 38 specific to the assembled virion subunit

The class II filamentous virus Pf3 packages a circular single-stranded DNA genome of approximately 5833 [corrected] nucleotides within a cylindrical capsid constructed from approximately 2500 [corrected] copies of a 44 residue alpha-helical subunit. The single tryptophan residue (Trp 38) of the capsid subunit is located within a basic C-terminal sequence (.R(+)WIK(+)AQFF). The local environment of Trp 38 in the native Pf3 assembly has been investigated using 229 nm excited ultraviolet-resonance Raman (UVRR) spectroscopy and fluorescence spectroscopy. Trp 38 exhibits an anomalous UVRR signature in Pf3, including structure-diagnostic Raman bands (763, 1228, 1370, and 1773 cm(-)(1)) that are greatly displaced from corresponding Raman markers observed in either detergent-disassembled Pf3, class I filamentous viruses, most globular proteins, or aqueous L-TRP. An unusual and highly quenched fluorescence spectrum is also observed for Trp 38. These distinctive UVRR and fluorescence signatures together reflect interactions of the Trp 38 side chain that are specific to the native PF3 assembly. The experimental results on PF3 and supporting spectroscopic data from other proteins of known three-dimensional structure favor a model in which pi electrons of the Trp 38 indolyl ring interact specifically with a basic side chain of the subunit C-terminal sequence. Residues Arg 37 AND Lys 40 are plausible candidates for the proposed cation-pi interaction of Trp 38. The present study suggests that raman spectroscopy may be a generally useful probe of interactions between the indolyl pi-electron system of tryptophan and electropositive groups in proteins and their assemblies.     

Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid

The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging. The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6. Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents. In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6. The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA. Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted. The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content. The fold of the core RNA polymerase (P2) is also mostly alpha-helical. On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type. Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid. These changes involve substantial ordering of the polypeptide backbone. Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2. An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex. Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.     

The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins

The complete amino acid sequence of echinoidin, the proposed name for a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina, has been determined by sequencing the peptides obtained from tryptic, Staphylococcus aureus V8 protease, chymotryptic, and thermolysin digestions. Echinoidin is a multimeric protein (Giga, Y., Sutoh, K., and Ikai, A. (1985) Biochemistry 24, 4461-4467) whose subunit consists of a total of 147 amino acid residues and one carbohydrate chain attached to Ser38. The molecular weight of the polypeptide without carbohydrate was calculated to be 16,671. Each polypeptide chain contains seven half-cystines, and six of them form three disulfide bonds in the single polypeptide chain (Cys3-Cys14, Cys31-Cys141, and Cys116-Cys132), while Cys2 is involved in an interpolypeptide disulfide linkage. From secondary structure prediction by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222) the protein appears to be rich in beta-sheet and beta-turn structures and poor in alpha-helical structure. The sequence of the COOH-terminal half of echinoidin is highly homologous to those of the COOH-terminal carbohydrate recognition portions of rat liver mannose-binding protein and several other hepatic lectins. This COOH-terminal region of echinoidin is also homologous to the central portion of the lectin from the flesh fly Sarcophaga peregrina. Moreover, echinoidin contains an Arg-Gly-Asp sequence which has been proposed to be a basic functional unit in cellular recognition proteins.     

Structural characterization of the filamentous bacteriophage PH75 from Thermus thermophilus by Raman and UV-resonance Raman spectroscopy

The filamentous bacteriophage PH75, which infects the thermophile T. thermophilus, assembles in vivo at 70 degrees C and is stable to at least 90 degrees C. Although a high-resolution structure of PH75 is not available, the virion is known to comprise a closed single-stranded (ss) DNA circle of 6500 nucleotides sheathed by a capsid comprising 2700 copies of a 46-residue subunit (pVIII). Here, we employ Raman and UV-resonance Raman (UVRR) spectroscopy to identify structural details of the pVIII and DNA constituents of PH75 that may be related to the high thermostability of the native virion assembly. Analysis of the Raman amide I and amide III signatures reveals that the capsid subunit secondary structure is predominantly (87%) alpha-helical but contains a significant number of residues (6 +/- 1 or 13 +/- 3%) differing from the canonical alpha-helix. This minor structural component is not apparent in capsid subunits of the mesophilic filamentous phages, fd, Pf1, and Pf3, previously examined at similar spectral resolution. The Raman signature of PH75 also differs from those of fd, Pf1, and Pf3 by virtue of an unusual alanine marker (898 cm(-)(1) band), which is attributed to C(alpha)-H hydrogen-bond donation by subunit Ala residues. Because alanines of the PH75 subunit occur primarily within sXXXs motifs (where s is a small side chain, e.g. Gly, Ala, Ser), and because the occurrence of such motifs in alpha-helices is believed to thermostabilize interhelix associations via C(alpha)-H...O interactions [G. Kleiger et al. (2002) Biochemistry 41, 5990-5997], we propose that such hydrogen bonding may explain both the alanyl and amide I/III markers of PH75 capsid subunits and that C(alpha)-H...O interactions may serve as a significant source of virion thermostabilization. Raman and UVRR signatures of PH75 are also distinguished from those of fd, Pf1, and Pf3 by several marker bands that are indicative of hydrophilic Trp and Tyr environments, including hydrogen bonding interactions of aromatic ring substituents. These interactions are likewise proposed as contributors to the high thermostability of PH75 vis-a-vis fd, Pf1, and Pf3. Finally, PH75 is the only filamentous phage exhibiting UVRR markers diagnostic of a highly base-stacked ssDNA genome incorporating the low energy C2'-endo/anti deoxynucleoside conformation. The present results suggest that both intersubunit interactions and genome organization contribute to the enhanced thermostability of PH75 relative to mesophilic filamentous bacteriophages.     

Intensity of the polarized Raman band at 1340-1345 cm-1 as an indicator of protein alpha-helix orientation: application to Pf1 filamentous virus

Raman spectra of oriented alpha-helical protein molecules exhibit a prominent band near 1340-1345 cm(-)(1), the intensity of which is highly sensitive to molecular orientation. Polarization of the 1340-1345 cm(-)(1) marker is evident in Raman spectra of alpha-helical poly-L-alanine (alphaPLA) and alpha-helical poly-gamma-benzyl-L-glutamate (alphaPBLG). Corresponding polarization is also observed in Raman spectra of the filamentous virus Pf1, which is an assembly of alpha-helical coat protein molecules. In alphaPLA and alphaPBLG, we assign the band to a normal mode of symmetry type E(2) and specifically to a vibration localized in the (O=C)-C(alpha)-H linkages of the main chain peptide group. Although strict helical symmetry does not apply to coat subunits of filamentous viruses, an approximate E(2)-type mode may be presumed to account for a corresponding Raman band of Pf1 and fd filamentous viruses. Spectroscopic studies of N-methylacetamide and isotopically-edited fd viruses support the present assignment of the 1340-1345 cm(-)(1) band. Polarization anisotropy indicates that this band may be exploited as a novel indicator of protein alpha-helix orientation. Application of this approach to the polarized Raman spectrum of Pf1 suggests that, on average, the axis of the alpha-helical coat protein subunit in the native virion structure forms an angle of 20 +/- 10 degrees with respect to the virion axis.     

Serine 171, a conserved residue in the gamma-aminobutyric acid type A (GABAA) receptor gamma2 subunit, mediates subunit interaction and cell surface localization

Serine 171 in the GABA(A) receptor gamma2 subunit is highly conserved in the ligand-gated ion channel superfamily. In this paper, we report that mutating serine 171 within gamma2 to glycine or cysteine prevents the interaction of gamma2 with alpha2 and beta1 when these subunits are co-expressed in human embryo kidney 293 cells, resulting in intracellular retention of gamma2. Structure analysis based on a three-dimensional homology model of gamma2 (Ernst, M., Brauchart, D., Boresch, S., and Sieghart, W. (2003) Neuroscience 119, 933-943) reveals that serine 171 may play a critical role in the formation and stabilization of an exposed turn structure that is part of the subunit interaction site. Mutation of serine 171 in the gamma2 subunit could therefore result in alteration of the structure of the subunit interaction site, preventing correct subunit assembly.     

Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution.

The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 Å resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit.The coenzyme binding domain of the subunit comprises residues 176 to 318. 45% of these residues are helical and 32% are in the central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit structure. The coenzyme analogue ADP-ribose binds to this domain in a position and orientation very similar to coenzyme binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket, the adenosine ribose is hydrogen-bonded to the side chain of Asp223, the pyrophosphate is positioned by interaction with Arg47 and the nicotinamide ribose is 6Å away from the catalytic zinc atom.The catalytic domain is mainly built up from three distinct antiparallel pleated-sheet regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. An approximate tetrahedral coordination of this zinc is completed by a water molecule or hydroxyl ion depending on the pH. Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cysteine residues 97, 100, 103 and 111. The lobe forms one side of a significant cleft in the enzyme surface suggesting that this region might constitute a second catalytic centre of unknown function.The two domains of the subunit are separated by a crevice that contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projecting out into the pocket. This water molecule is hydrogen-bonded to the side chain of Ser48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and the nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Both subunits contribute residues to each of the two substrate binding pockets of the molecule. The only accessible polar groups in the vicinity of the catalytic centre are Ser48 and Thr178 apart from zinc and the zinc-bound water molecule.     

Structural studies of bean pod mottle virus, capsid, and RNA in crystal and solution states by laser Raman spectroscopy

Structures of protein and RNA components of bean pod mottle virus (BPMV) have been investigated by use of laser Raman spectroscopy. Raman spectra were collected from both aqueous solutions and single crystals of BPMV capsids (top component) and virions (middle and bottom components, which package, respectively, small and large RNA molecules). Analysis of the data permits the assignment of conformation-sensitive Raman bands to viral protein and RNA constituents and observation of structural similarities and differences between solution and crystalline states of BPMV components. The Raman results show that the protein subunits of the empty capsid contain between 45% and 55% beta-strand and beta-turn secondary structure, in agreement with the recently determined X-ray crystal structure, and that this total beta-strand content undergoes a small increase (approximately 5%) with packaging of RNA. The subunits are relatively deficient in alpha-helix secondary structure, estimated at less than 25%, and therefore must contain extensive amounts (greater than 20%) of loops and irregular chain conformations. The Raman spectra also show the following: (1) The molecular environments of as many as four tryptophan residues per subunit are altered upon packaging RNA, resulting in stronger 1N-H hydrogen bonding for two Trp residues and more hydrophobic environments for two indole rings. (2) Hydrogen-bonding states of the seven Tyr residues per subunit do not change detectably when RNA is packaged. At least five tyrosine OH groups are involved exclusively as strong hydrogen bond donors to protein acceptor groups, which suggests restricted access of solvent H2O molecules to these parahydroxyls.(ABSTRACT TRUNCATED AT 250 WORDS)     

The intact human acetylcholinesterase C-terminal oligomerization domain is alpha-helical in situ and in isolation,but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers

A 14-residue fragment of the C-terminal oligomerization domain, or T-peptide, of human acetylcholinesterase (AChE) shares sequence homology with the amyloid-beta peptide implicated in Alzheimer's disease and can spontaneously self-assemble into classical amyloid fibrils under physiological conditions [Greenfield, S. A., and Vaux, D. J. (2002) Neuroscience 113, 485-492; Cottingham, M. G., Hollinshead, M. S., and Vaux, D. J. (2002) Biochemistry 41, 13539-13547]. Here we demonstrate that the conformation of this AChE(586-599) peptide, both before and after fibril formation, is different from that of a longer peptide, T(40), corresponding to the entire 40-amino acid T-peptide (residues 575-614 of AChE). This peptide is prone to homomeric hydrophobic interactions, consistent with its role in AChE subunit assembly, and possesses an alpha-helical structure which protects against the development of the beta-sheet-rich amyloidogenic conformation favored by the shorter constituent AChE(586-599) fragment. Using a conformation-sensitive monoclonal antibody raised against the alpha-helical T(40) peptide, we demonstrate that the conformation of the T-peptide domain within intact AChE is antigenically indistinguishable from that of the synthetic T(40) peptide. A second monoclonal antibody raised against the fibrillogenic AChE(586-599) fragment recognizes not only beta-sheet amyloid aggregates but also SDS-resistant protofibrillar oligomers. A single-antibody sandwich ELISA confirms that such oligomers exist at micromolar peptide concentrations, well below that required for formation of classical amyloid fibrils. Epitope mapping with this monoclonal antibody identifies a region near the N-terminus of the peptide that remains accessible in oligomer and fibril alike, suggesting a model for the arrangement of subunits within AChE(586-599) protofibrils and fibrils.