Theoretical Studies toward Quantitative Protein Circular Dichroism Calculations

Besley, Nicholas A.; Hirst, Jonathan D.

doi:10.1021/ja990627l

Cited by 161 publications

(231 citation statements)

References 77 publications

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“…2a). One possible explanation for this band is that the serine residue disrupts the normal ␤-sheet structure of the protein, leading to the formation of a ''␤ -bulge'' (15,16). Should such a bulge form, the closest tryptophan (Trp-42) would become more solvent-exposed, resulting in a redshift of the emission maximum for the tryptophan fluorescence (14,17).…”

Section: Resultsmentioning

confidence: 99%

Crystal cataracts: Human genetic cataract caused by protein crystallization

Pande

Pande²,

Asherie³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

208

207

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Several human genetic cataracts have been linked recently to point mutations in the ␥D crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of ␥D crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.H uman ␥D crystallin is a member of a highly homologous family of mammalian lens proteins called the ␥ crystallins (1). Together with the ␣ and ␤ crystallins, these proteins are essential for maintaining lens transparency. However, the ␥ crystallins differ from the ␣ and ␤ crystallins in one important respect: the interactions between the ␥ crystallins are attractive (2). This feature reduces the osmotic pressure in the lens, but it also makes the ␥ crystallins more susceptible to aggregation and phase separation, phenomena that diminish the homogeneity of the lens and lead to cataract (3). Yet, despite these attractive interactions, the ␥ crystallins remain soluble for many years at high concentrations and with little turnover, maintaining the proper refractive index gradient of the lens (4).It is well known that random mutations in proteins dramatically affect their solubility (5). In this article, we show that the Arg-58 to His (R58H) mutant [linked to the aculeiform cataract (Fig. 1a; ref. 6)] and the Arg-36 to Ser (R36S) mutant [linked to another form of genetic (congenital) cataract ( Fig. 1b; ref. 7)] are much less soluble than the wild-type human ␥D crystallin protein (HGD). We also show that these mutants are more prone to crystallize than the wild-type. Indeed, Kmoch et al. (7) recently extracted crystals of the R36S mutant from the eye of a young patient. To determine the mechanism of cataract formation caused by these mutations, we compared the conformation, stability, and phase behavior of the recombinant HGD, R58H, and R36S proteins in solution. Materials and MethodsCloning, Expression, and Isolation of Proteins. Recombinant human ␥D crystallin was prepared by the amplification of the coding sequence from a human fetal lens cDNA library as detailed (8). Overexpression of ␥D crystallin, and isolation and purification of the protein, were all done according to procedures as described (8). Mutant proteins were prepared as follows.(i) The R58H mutant. To introduce a histidine mutation in place of Arg-58, the following oligonucleotide primers were made: 5Ј-CCAGTACTTCCTGCACCGCGGCGACTATGC-3Ј as the forward primer and 5Ј-GCATAGTCGCCGCGGTGCAG-GAAGTACTGG-3Ј as the reverse prime...

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Section: Resultsmentioning

confidence: 99%

Crystal cataracts: Human genetic cataract caused by protein crystallization

Pande

Pande²,

Asherie³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

208

207

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“…29,32 Monopoles for each transition between electronic states were determined by fitting their electrostatic potential to reproduce the ab initio electrostatic potential for that transition so that the least-squares difference is minimized (typically within 5%). The parameter set 24 consists of 32 monopoles for the amide n f π* transition all at a distance of 0.1 Å from the C, N, O, and H atoms (forming a cube around each atom), and 20 monopoles for the π f π* transition, with one charge situated at each atom center and four around each center at a distance of 0.05 Å (all in the peptide plane). To calculate the electric and magnetic transition dipole moments of electronic transitions in proteins, the matrix method [33][34][35][36] is commonly used.…”

Section: Theoretical Methodsmentioning

confidence: 99%

Charge-Transfer Transitions in the Vacuum-Ultraviolet of Protein Circular Dichroism Spectra

et al. 2008

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Circular dichroism (CD) is widely used in the structural characterization and secondary structure determination of proteins. The vacuum UV region (below 190 nm), where charge-transfer transitions have an influence on the CD spectra, can be accessed using synchrotron radiation circular dichroism (SRCD) spectroscopy. Recently, charge-transfer transitions in a conformationally diverse set of dipeptides have been characterized ab initio using complete active space self-consistent field calculations, and the relevant charge distributions have been parametrized for use in the matrix method for calculations of protein CD. Here, we present calculations of the vacuum UV CD spectra of 71 proteins, for which experimental SRCD spectra and X-ray crystal structures are available. The theoretical spectra are calculated considering charge-transfer and side chain transitions. This significantly improves the agreement with experiment, raising the Spearman correlation coefficient between the calculated and the experimental intensity at 175 nm from 0.12 to 0.79. The influence of the conformation on charge-transfer transitions is analyzed in detail, showing that the n f π* charge-transfer transitions are most important in R-helical proteins, whereas in strand proteins the π f π* charge-transfer transition along the chain in the amino-to carboxy-end direction is most dominant.

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“…2A). Although these observations indicate that there is not a substantial change in the net secondary-structure content of the protein, some change in local secondary structure remains possible due to complexities in the spectral properties of short ␤-strand structures (59). CD shows that thermal unfolding proceeds via a single transition (Fig.…”

Section: Conformational Change In Alkbmentioning

confidence: 96%

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

Ergel

Gill

Brown

et al. 2014

Journal of Biological Chemistry

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Background: AlkB is an iron/2-oxoglutarate dioxygenase that repairs alkylated DNA. Results: Enzymological and biophysical methods are used to develop a quantitative mechanistic scheme. Conclusion: A conformational transition controls the order of substrate binding and the coupling of the two sequential sub-reactions catalyzed by AlkB. Significance: These results provide a striking example of the importance of protein dynamics for efficient catalysis of a multistep reaction.

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Theoretical Studies toward Quantitative Protein Circular Dichroism Calculations

Cited by 161 publications

References 77 publications

Crystal cataracts: Human genetic cataract caused by protein crystallization

Crystal cataracts: Human genetic cataract caused by protein crystallization

Charge-Transfer Transitions in the Vacuum-Ultraviolet of Protein Circular Dichroism Spectra

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

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