Derivative Absorbance Spectroscopy and Protein Phase Diagrams as Tools for Comprehensive Protein Characterization: A bGCSF Case Study

Kueltzo, Lisa A.; Ersoy, Baran A.; Ralston, John P.; Middaugh, C. Russell

doi:10.1002/jps.10439

Cited by 120 publications

(169 citation statements)

References 16 publications

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“…no reversibility is implied), but rather simple, empirical representations of the physical behavior of the macromolecule. [1][2][3] As shown in Fig-ure 5(B), a small pink-colored region between the native and unfolded state is also observed in the three-index EPD. The calculated structural indices used for the construction of the three-index EPD for the protein antigen SP1732 are shown in Figure 6(G-I; see Methods section).…”

Section: Resultsmentioning

confidence: 69%

“…This includes the lack of meaningful relationship between color itself and actual molecular features (in EPDs, structural changes are represented by color transitions 1,2,9 ) as well as limitations resulting from color deficiencies in vision and blindness, which are possessed by a substantial portion of the human population. Red-green color vision defects are found in about 8% of males and 0.5% of females among people of Northern European origin, 5% among Chinese and Japanese populations, and 4% or less among individuals of African origin.…”

Section: -10mentioning

confidence: 99%

“…Using singular value decomposition (SVD), the three largest contributions to a vector are obtained and reduced to color based on an RGB scheme. 1,2 Although the actual colors are somewhat arbitrary, changes in color are useful, because they represent structural changes. In addition, differences between EPDs can be directly compared (e.g., between mutants of the same protein) by processing all the data together.…”

Section: Introductionmentioning

confidence: 99%

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Improved data visualization techniques for analyzing macromolecule structural changes

et al. 2012

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The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color-blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three-index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.

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Section: Resultsmentioning

confidence: 69%

Section: -10mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improved data visualization techniques for analyzing macromolecule structural changes

et al. 2012

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“…This is particularly useful for macromolecular complexes displaying aggregation such as RSV. 30,31 In general, when aromatic amino acid side chains are moved to a more polar environment, this causes a blue shift in the absorbance peaks, while shifts to longer red wavelengths indicate that the residues are in a more buried or less polar location. 30 Six major negative peaks were resolved within the absorbing regions of the aromatic residues in the second derivative UV spectrum of RSV (spectra not shown).…”

Section: High Throughput Screening For Inhibitors Of Rsv Aggregationmentioning

confidence: 99%

High-throughput screening of stabilizers for Respiratory Syncytial Virus: Identification of stabilizers and their effects on the conformational thermostability of viral particles

Ausar

Espina²,

Brock³

et al. 2007

Human Vaccines

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Respiratory syncytial virus (RSV) is the leading cause of severe respiratory infection in children worldwide. Recombinant live attenuated viral preparations are one of the most promising strategies for vaccination but they typically possess poor thermostability. In this work, a library of compounds was screened and stabilizers were selected based on their ability to inhibit the aggregation of RSV perturbed at 56˚C. After screening and selection of excipients, the conformational stability of the RSV proteins was evaluated in the presence of potential stabilizers. The secondary and tertiary structures as well as aggregation/dissociation of RSV were monitored using circular dichroism and second derivative UV absorption spectroscopies and light scattering, respectively, as a function of temperature (10-90˚C). RSV membrane fluidity was also evaluated by generalized polarization of Laurdan fluorescence. Screening experiments showed that a variety of sugars, amino acids, polyols and polyanions inhibited the aggregation of viral particles. Conformational stability studies demonstrated that the addition of sugars and polyols stabilized RSV as indicated by a significant increase in the transition melting temperature (Tm) of both the secondary and tertiary structures as well as the gel to liquid crystalline membrane transition. These results should provide the basis for rational development of more physically stable formulations of live attenuated RSV vaccines.

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“…In this work we have examined the effects of pH and temperature on intact NV-VLPs using a combination of several spectroscopic methods as well as calorimetric and microscopic analyses. Synthesis of the spectroscopic data, employing a multidimensional eigenvector phase space approach (14), provides a comprehensive description of the physical stability of the NV-VLP. Complementary, calorimetric analysis revealed that the two different domains of VP1 unfold independently in a pH-dependent manner.…”

mentioning

confidence: 99%

Conformational Stability and Disassembly of Norwalk Virus-like Particles

Ausar

Foubert²,

Hudson³

et al. 2006

Journal of Biological Chemistry

214

161

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Greater than 99% of the Norwalk virus (NV) capsid consists of 180 copies of a single 58-kDa protein. Recombinantly expressed monomers self-assemble into virus-like particles (VLPs) with a well defined icosahedral structure. NV-VLPs are an appropriate vaccine antigen since the antigenic determinants of the parent virion are preserved. They also constitute very simple models to study the mechanisms of assembly and disassembly of viral capsids. This work examines the inherent stability of NV-VLPs over a range of pH and temperature values and provides detailed insight into structural perturbations that accompany disassembly. The NV-VLP structure was monitored using a variety of biophysical techniques including intrinsic and extrinsic fluorescence, high resolution second-derivative UV absorption spectroscopy, circular dichroism (CD), dynamic light scattering, differential scanning calorimetry, and direct observation employing transmission electron microscopy. The data demonstrate that NVVLPs are highly stable over a pH range of 3-7 and up to 55°C. At pH 8, however, reversible capsid dissociation was correlated with increased solvent exposure of tyrosine residues and subtle changes in secondary structure. Above 60°C NV-VLPs undergo distinct phase transitions arising from secondary-, tertiary-, and quaternary-level protein structural perturbations. By combining the spectroscopic data employing a multidimensional eigenvector phase space approach, an empirical phase diagram for NV-VLP was constructed. This strategy of visualization provides a comprehensive description of the physical stability of NV-VLP over a broad range of pH and temperature. Complementary, differential scanning calorimetric analyses suggest that the two domains of VP1 unfold independently in a pH-dependent manner. Norwalk virus (NV)2 and Norwalk-like viruses are responsible for more than 95% of acute nonbacterial gastroenteritis with 23 million reported cases per year in the United States alone (1). These viruses are members of the family Caliciviridae and the recently designated genus Norovirus, which comprises genogroups I and II. They are highly contagious, requiring low numbers of viruses for infection and appear to be stable outside the host for several days. Transmission of NV occurs primarily via the fecal-oral route with several outbreaks of waterborne and food-borne gastroenteritis documented on cruise ships, in schools, nursing homes, residential institutions, and areas devastated by natural disasters (1, 2). NV outbreaks have also been documented in the United States military population and in different areas of the world where crowded living conditions facilitated person to person spread (3, 4). NV and other members of the Caliciviridae family have recently been designated as category B biodefense pathogens (National Institutes of Health Publication 03-5315). The gastroenteritis associated with NV has an average incubation period of 12-48 h, with symptoms characterized by acute onset of nausea, vomiting, and diarrhea. Although the illness ...

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Derivative Absorbance Spectroscopy and Protein Phase Diagrams as Tools for Comprehensive Protein Characterization: A bGCSF Case Study

Cited by 120 publications

References 16 publications

Improved data visualization techniques for analyzing macromolecule structural changes

Improved data visualization techniques for analyzing macromolecule structural changes

High-throughput screening of stabilizers for Respiratory Syncytial Virus: Identification of stabilizers and their effects on the conformational thermostability of viral particles

Conformational Stability and Disassembly of Norwalk Virus-like Particles

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