Marc S. Lewis

Modern analytical ultracentrifugation in protein science: A tutorial review

Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self‐association and heterogeneous interactions, such as protein–protein, protein–nucleic acid, and protein–small molecule binding.

The incorporation of [14C]glycine into the subunits of collagens from normal and lathyritic animals

Abstract The incorporation of [ 14 C]glycine into the subunits of skin collagen was followed in normal and lathyritic rats. Label appears first and at the same rate in the primary subunits, α1 and α2, and later in β1 and β2. This is explained by the fact that β1 and β2 are derived from α1 and α2 by intramolecular crosslinking. In lathyritic animals the formation of β1 and β2 is markedly reduced though the synthesis of collagen proceeds. This defect is evident long before the appearance of the gross symptoms of lathyrism. The results are consistent with the suggestion that the lack of intramolecular crosslinks affects the integrity of connective tissue through a loss of fibril stability resulting in the gross symptoms characteristic of lathyrism.

Differential binding to soluble nuclear receptors and effects on cell viability of retinol and retinoic acid in cultured retinoblastoma cells.

Abstract Human retinoblastoma cells in culture contain soluble receptors for both 3H-retinol and 3H-retinoic acid which are separate and distinct as assessed by specificity, enzyme susceptibility and binding affinity. Total cellular binding of retinol correlates well with its rapid effect on cell mortality. A limited number of soluble nuclear receptor sites for 3H-retinoic acid but not 3H-retinol are observed after incubation of the 3H-retinoid with intact cells indicating a possible preferential effect of retinoic acid at the gene level.

Tropomyosin, myosin and actin from the blowfly, Phormia regina

Abstract 1. 1. Purified tropomyosin has been prepared from adult and larval stages of the blowfly, Phormia regina ; purified myosin and actin have been prepared from the adult stage only. Special procedures were required in the preparation of all three proteins. 2. 2. The amino acid compositions of these four purified proteins have been determined and compared to those of the corresponding rabbit proteins. The charged residues of Phormia tropomyosins are the same as those of lobster and most molluscs. Although there is no significant difference in amino acid composition between the two insect tropomyosins, fingerprint patterns of their tryptic digests suggest that structural differences may exist. 3. 3. The sedimentation rate, molecular weight, and intrinsic viscosity of adult Phormia tropomyosin are less than those of the more polymerizable larval protein. The adult values are: S 20 0 = 2.53, M 0 = 65 600, [ η ] = 0.23. The sedimentation rate and intrinsic viscosity of rabbit skeletal tropomyosin have been found to be comparable to those of the Phormia tropomyosins. 4. 4. Optical-rotary-dispersion studies show 100% ∞-helical configuration of the two insect tropomyosins, and the hydrodynamic measurements favor a 2-stranded structure.

VITAMIN A RECEPTORS: RETINOL BINDING IN NEURAL RETINA AND PIGMENT EPITHELIUM

Abstract— With sucrose density gradient analysis, bovine retinal cytosol demonstrates 2S and 7S retinol‐binding species; binding in pigment epithelial cytosol is predominantly to a 2S species. Binding of retinol to the 2S component in retina is unaffected by retinoic acid or retinyl palmitate whereas the ester effectively competes for 2S binding in pigment epithelium. Specific retinol binding can also be demonstrated by gel filtration on Sepharose 4B; no high molecular weight (> 100–200,000) retinol binding species are observed by this technique. Both the 2S and 7S binding species in retinal cytosol are protein in nature and differentially susceptible to proteolysis. The 2S and 7S species appear to be separate and distinct since chaotropic agents such as sodium thiocyanate or KCl and CaCl2 do not seem to convert the 7S species into 2S subunits. Scatchard plot analysis indicates high affinity retinol binding to the 2S receptor. Computer analysis of the binding data yields Ka=3 × 108, n = 1, a molecular size of 16,200 and ΔG0=−9.5 kcal/mol.

Inactivation of DNA Mismatch Repair by Increased Expression of Yeast MLH1

ABSTRACT Inactivation of DNA mismatch repair by mutation or by transcriptional silencing of the MLH1 gene results in genome instability and cancer predisposition. We recently found (P. V. Shcherbakova and T. A. Kunkel, Mol. Cell. Biol. 19:3177–3183, 1999) that an elevated spontaneous mutation rate can also result from increased expression of yeast MLH1. Here we investigate the mechanism of this mutator effect. Hybridization of poly(A)+ mRNA to DNA microarrays containing 96.4% of yeast open reading frames revealed that MLH1overexpression did not induce changes in expression of other genes involved in DNA replication or repair. MLH1overexpression strongly enhanced spontaneous mutagenesis in yeast strains with defects in the 3′→5′ exonuclease activity of replicative DNA polymerases δ and ɛ but did not enhance the mutation rate in strains with deletions of MSH2, MLH1, orPMS1. This suggests that overexpression ofMLH1 inactivates mismatch repair of replication errors. Overexpression of the PMS1 gene alone caused a moderate increase in the mutation rate and strongly suppressed the mutator effect caused by MLH1 overexpression. The mutator effect was also reduced by a missense mutation in the MLH1 gene that disrupted Mlh1p-Pms1p interaction. Analytical ultracentrifugation experiments showed that purified Mlh1p forms a homodimer in solution, albeit with a K d of 3.14 μM, 36-fold higher than that for Mlh1p-Pms1p heterodimerization. These observations suggest that the mismatch repair defect in cells overexpressingMLH1 results from an imbalance in the levels of Mlh1p and Pms1p and that this imbalance might lead to formation of nonfunctional mismatch repair complexes containing Mlh1p homodimers.

Improving the Thermal, Radial and Temporal Accuracy of the Analytical Ultracentrifuge through External References

Sedimentation velocity (SV) is a method based on first principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton temperature logger to directly measure the temperature of a spinning rotor and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration that were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., Anal. Biochem., 437 (2013) 104-108), and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from 11 instruments displayed a significantly reduced standard deviation of approximately 0.7%. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.

Retinol receptors in corneal epithelium, stroma and endothelium

Specific receptors for retinol are present in the cytosol fraction of corneal epithelium as demonstrated by sucrose density gradient centrifugation. These appear to be (1) protein in nature (2) of small molecular size (2 S) (3) specific for retinol and (4) present in several species. Assuming a receptor molecular weight of 15 000 and a single mole of retinol bound/mole of receptor protein, the association constant value is 5.26-10(7) with deltaG degrees = -8.53 kcal/mol. 2-S receptors are also observed in stroma and endothelium along with another binding species of approximately 8 S. Binding of [3H]retinol in bovine epithelial cytosol can also be demonstrated by disc gel electrophoresis and gel filtration. Immunodiffusion techniques demonstrate that monkey corneal epithelial and stromal cytosol samples do not contain contaminating serum retinol binding-protein.

The molecular weight and detergent binding of bovine rhodopsin.

Abstract The molecular weight and the Triton X-100 binding of bovine rhodopsin have been measured by means of analytical ultracentrifugation. A molecular weight of 35 000 was found for the delipidated protein. Binding studies indicated a maximum of 362 sites and the intrinsic association constant gave a free energy of binding of −4·52 kcal/mol Triton X-100 bound, as compared with a free energy for detergent self association of −3·52 kcal/mol monomer. Studies on the detergent concentration dependence of rhodopsin extraction permitted estimation of the maximum total free energy membrane lipid binding to rhodopsin as −406 kcal/mol rhodopsin. Knowledge of the maximum number of binding sites made possible an estimate of the molecular surface area, and this parameter, combined with the molecular volume permitted the calculation of the molecular dimensions for various models.

Self-association in highly concentrated solutions of myoglobin: a novel analysis of sedimentation equilibrium of highly nonideal solutions.

Abstract The sedimentation equilibrium in concentrated solutions of hemoglobin and myoglobin has been measured. The ratio of the apparent molecular weight of hemoglobin to that of myoglobin. R . is found to obey the empirical relation R ( c ) = 3.8–4.25 × 10 −2 c +6.44 × 10 −4 c 2 − 2.21 × 10 −5 c 3 , where c is the protein concentration in g/dl. for c −⩽40 g/dl. A theoretical relation for the dependence of R upon c in the absence of protein self-association is presented. This relation cannot be fitted to the experimental results, and the discrepancy is attributed to self-association of myoglobin.