Abstract:SUMMARYAn early step in the morphogenesis of the double-stranded DNA bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a… Show more
“…2B-E). The modeling was based on the secondary-structure predictions and biophysical analyses (Benevides et al, 2004; Němeček et al, 2009) as the limited resolution of the reconstruction within the capsid interior precludes distinguishing individual α-helices. A marked deviation from quasi-equivalence was observed in the organization of the scaffolding domains with helices belonging to domains A/D and C/F being respectively the best and worst defined in the density.…”
Most dsDNA viruses, including bacteriophages and herpesviruses, rely on a staged assembly process of capsid formation. A viral protease is required for many of them to disconnect scaffolding domains/proteins from the capsid shell, therefore priming the maturation process. We used the bacteriophage HK97 as a model system to decipher the molecular mechanisms underlying the recruitment of the maturation protease by the assembling procapsid and the influence exerted onto the latter. Comparisons of the procapsid with and without protease using single-particle electron cryomicroscopy reconstructions, hydrogen/deuterium exchange coupled to mass spectrometry and native mass spectrometry demonstrated that the protease interacts with the scaffolding domains within the procapsid interior and stabilizes them as well as the whole particle. The results suggest that the thermodynamic consequences of protease packaging are to shift the equilibrium between isolated coat subunit capsomers and procapsid in favor of the latter by stabilizing the assembled particle before making the process irreversible through proteolysis of the scaffolding domains.
“…2B-E). The modeling was based on the secondary-structure predictions and biophysical analyses (Benevides et al, 2004; Němeček et al, 2009) as the limited resolution of the reconstruction within the capsid interior precludes distinguishing individual α-helices. A marked deviation from quasi-equivalence was observed in the organization of the scaffolding domains with helices belonging to domains A/D and C/F being respectively the best and worst defined in the density.…”
Most dsDNA viruses, including bacteriophages and herpesviruses, rely on a staged assembly process of capsid formation. A viral protease is required for many of them to disconnect scaffolding domains/proteins from the capsid shell, therefore priming the maturation process. We used the bacteriophage HK97 as a model system to decipher the molecular mechanisms underlying the recruitment of the maturation protease by the assembling procapsid and the influence exerted onto the latter. Comparisons of the procapsid with and without protease using single-particle electron cryomicroscopy reconstructions, hydrogen/deuterium exchange coupled to mass spectrometry and native mass spectrometry demonstrated that the protease interacts with the scaffolding domains within the procapsid interior and stabilizes them as well as the whole particle. The results suggest that the thermodynamic consequences of protease packaging are to shift the equilibrium between isolated coat subunit capsomers and procapsid in favor of the latter by stabilizing the assembled particle before making the process irreversible through proteolysis of the scaffolding domains.
“…The SVD approach is illustrated in Figure for a series of temperature‐dependent spectra of the in vitro–purified Δ‐domain of the bacteriophage HK97 coat protein in the temperature interval of 10° to 90°C (Nemecek et al, ). The whole spectral interval of 580 to 1780 cm −1 (1200 spectral points) was analyzed and the results include concurrent changes including markers of the main peptide chain secondary structure—the amide III (1210 to 1310 cm −1 ) and amide I (1640 to 1680 cm −1 ) bands—as well as markers of individual side chains—Tyr (820 to 840 cm −1 ), Phe (1003 cm −1 ), and Trp (1340 to 1360 cm −1 ) bands.…”
Section: Discussionmentioning
confidence: 99%
“…The gray lines show the fit of V i1 ‐V i4 coefficients. Data are adapted with permission from Nemecek et al ()…”
Section: Applications To Assemblies Of Viral Proteinsmentioning
A protein Raman spectrum comprises discrete bands representing vibrational modes of the peptide backbone and its side chains. The spectral positions, intensities, and polarizations of the Raman bands are sensitive to protein secondary, tertiary, and quaternary structures and to side-chain orientations and local environments. In favorable cases, the Raman spectrum serves as an empirical signature of protein three-dimensional structure, intramolecular dynamics, and intermolecular interactions. Quantitative analysis of Raman spectral series can be further boosted by advanced statistical approaches of factor analysis that allow fitting of specific theoretical models while reducing the amount of analyzed data. Here, the strengths of Raman spectroscopy are illustrated by considering recent applications from the authors' work that address (1) subunit folding and recognition in assembly of the icosahedral bacteriophages, (2) orientations of subunit main chains and side chains in native filamentous viruses, (3) roles of cysteine hydrogen bonding in the folding, assembly, and function of virus structural proteins, and (4) structural determinants of protein/DNA recognition in gene regulatory complexes. Conventional Raman and polarized Raman techniques are surveyed.
“…Instead of looking for and presenting obvious spectral differences between a few selected Raman spectra taken under definite conditions, more complex and less apparent spectral changes can be discerned, studied and interpreted as a function of a particular physicochemical parameter (intensive variable [1] ), gradually varied over a given range. Thermal-induced structural transitions of biomolecules, [2,3] acid-base titrations [4,5] or kinetic studies [6,7] monitored by Raman spectroscopy can be taken as an archetype of particular but frequent Raman experiments when a biological analyte is dissolved in an aqueous buffer to rather low concentration, and spectral changes are studied as a function of temperature, pH and time, respectively. Instead of laborious visual inspection and manual identification of spectral changes, extensive series of Raman spectra can be analyzed by multivariate statistical methods [8] (factor analysis, principal component analysis).…”
Section: Introductionmentioning
confidence: 99%
“…solvent peaks or particular Raman bands of the analyte itself, [2,11] or bands of a foreign compound added to the sample ad hoc, e.g. ClO 4 − , NO 3 − , SO 4 2− , SeO 4 2− or PO 4 2− ions. [11,12] In both cases, an ideal internal standard should provide intense, clearly resolvable Raman features not coinciding with other Raman bands and remaining unaffected by the physicochemical parameter varied over the entire range of interest.…”
A semiautomated method combining intensity normalization with effective elimination of the solvent signal and non-Raman background is presented for Raman spectra of biochemical and biological analytes in aqueous solutions. The method is particularly suitable for rapid and effortless preprocessing of extensive datasets taken as a function of gradually varied physicochemical parameters, e.g. analyte and/or ligand concentration, temperature, pH, pressure, ionic strength, time, etc. For intensity normalization, the strong Raman OH stretching band of water in the range of 2700-3900 cm −1 recorded together with the analyte spectrum in the fingerprint region below 1800 cm −1 is employed as internal intensity standard. Concomitant dependences of the solvent Raman spectra are taken into account and, in some cases, turned into advantage. Once the Raman spectra of the solvent are acquired for a particular range of the parameter varied, solvent contribution can be subtracted correctly from any analyte spectrum taken within this range. The procedure presented can be efficiently applied only for the analytes having their own Raman signal in the range of OH stretching vibrations much weaker than that of the solvent. However, this is the case for a great number of biochemical and biological samples. Accuracy, reliability and robustness of the method were tested under the conditions of spontaneous Raman, resonance Raman and surface-enhanced Raman scattering.
Serviceability of the method is demonstrated by several real-world examples.
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