Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris.

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.

Numerical study of the Johnston-Ogston effect in two-component systems.

Numerical solutions of the Lamm equation are presented for systems exhibiting the Johnston--Ogston effect. From these solutions it is apparent that the movement of the maxima of the concentration gradient curves reflects the sedimentation velocity of the slow or fast components in their appropriate plateaus. A simple generalization of the Johnston--Ogston analysis is presented, valid for all centrifugation times in a radial field and sector shaped cell provided only that there exist both a plateau of the slow component by itself and the mixed plateau with both slow and fast components present.

Subunit association and heterogeneity of Limulus polyphemus hemocyanin.

The molecular weights of the 6S, 24S, 36S, and 60S components of Limulus polyphemus hemocyanin were determined by high speed sedimentation equilibrium to be 69 400, 856 000, 1 690 000, and 3 160 000. The behavior of this hemocyanin appears to be similar to that of other arthropod hemocyanins where the first aggregation step is the formation of a hexamer of the 6S monomer. Here the larger aggregated states (24S, 36S, and 60S) are successive dimers of an unobserved hexamer (16S). The 24S-36S-60S association was found to be heterogeneous, suggesting that 24S components of different composition may be present.

An extrapolation method for reducing equilibration times in sedimentation equilibrium experiments.

We present a detailed investigation of the use of an extrapolation technique to decrease running times of sedimentation equilibrium experiments. If concentration profiles are available at time delta tau, 2delta tau, 3delta tau,...., cn(r) = c(r, n delta tau), then the Aitken transformation replaces the cn(r) + cn(r) = [cn + 1(r) cn - 1(r) - c2n(r)]/[cn + 1(r) + cn - 1(r) - 2cn(r)]. We show that the cn(r) converge to the equilibrium values c infinity (r) much more quickly than the cn(r). Savings in time are shown to range from a factor of approximately 2 for meniscus depletion experiments to factors of between 4 and 8 for lower speeds or smaller molecular weights. It is also shown that the technique is quite sensitive to noise, so that an accurate optical system is required to allow its optimal use.

Molecular weight of recombinant human tumor necrosis factor-alpha.

Recombinant DNA-derived human tumor necrosis factor-alpha from Escherichia coli was examined by equilibrium ultracentrifugation under conditions similar to those where gel filtration experiments suggested an oligomeric structure. Short-column equilibrium experiments at concentrations in the range 0.015-0.12% at pH 8.5 in 0.04 M Tris/Tris-HCl gave molecular weights corresponding to 3 times the sequence molecular weight both in the presence and absence of 0.1 M NaCl. Long (2.6 mm)-column experiments under the same solvent conditions indicated molecular weights of 51,900 +/- 900 in the absence of added NaCl and 52,600 +/- 700 in the presence of added 0.1 M NaCl. No evidence of any species other than the trimer was found.

Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma.

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.

Virial expansions for ideal self-associating systems.

Theoretical virial equations for self-associating systems governed by mass action have been derived assuming the solute to be ideal except for this solutesolute interaction. In particular, monomer-polymer association involving two molecular species and isodesmic association involving an indefinite number of molecular species have been treated analytically. The usefulness of such virial equations is severely limited by their extremely narrow interval of convergence.

Effect of some proteins on the yeast cell membrane.

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.

Rapid precision interferometry for the analytical ultracentrifuge. III. Determination of period of rotation, frequency of rotation, and elapsed time.

The approaches presented in this series of papers make possible rapid gathering, reduction, and analysis of data from the Rayleigh interference optical system of an analytical ultracentrifuge. Instrumentation described in this paper provides some of the timing and measurement circuits necessary for a microprocessor or minicomputer to determine the rotor frequency, rotor period, and elapsed time of an experiment. It includes simple but effective circuits to generate precise rotor timing pulses that are useful for synchronization of pulsed light sources. Circuits to control photographic operations in the ultracentrifuge are described briefly. All of these circuits are interfaced to a simple microcomputer address/data bus. An adapter between this bus and a Q-bus (for a DEC LSI-11/2 or LSI 11/23 microcomputer) is also described. The circuits presented have been used in this laboratory over a 3-year period. They have proven reliable and form an integral part of the real-time data acquisition systems that have been constructed.

Rapid precision interferometry for the analytical ultracentrifuge. II. A laser controller based on a rate-multiplying circuit.

A laser controller that uses a fixed frequency clock and a digital rate-multiplying circuit to synchronize the triggering of a pulsed laser to the spinning of an analytical ultracentrifuge rotor has been designed. The circuit is simple, inexpensive, and virtually free of any adjustments. It tracks rotors undergoing full acceleration or deceleration. At constant rotor speed it provides triggering that is accurate and reproducible to better than 0.5 microseconds. The settings of this controller are independent of rotor speed over the full range of the ultracentrifuge.

Rapid precision interferometry for the analytical ultracentrifuge. I. A laser controller based on a phase-lock-loop circuit.

This is the first of a series of manuscripts presenting methods to enable rapid reduction of data from the Rayleigh interference optical system of the Beckman Model E analytical ultracentrifuge. Here we present a pulsed laser controller for the ultracentrifuge. This laser controller uses a phase-lock-loop to provide properly timed light pulses over the speed range of 3000 to 60,000 rpm; it effectively resolves one rotor revolution into 4096 discrete angular positions. The circuit has been designed so that the laser light bursts occur at selectable angular positions of the rotor that are independent of rotor speed even under conditions of maximum acceleration or deceleration. We have used this controller in our laboratory over a 7-year period for both photographic and real-time collection at interferometric data from the ultracentrifuge.

Acid unfolding and self-association of recombinant Escherichia coli derived human interferon gamma.

The secondary and tertiary structure of recombinant human interferon gamma, determined by far- and near-UV circular dichroism, showed a transition from the native state to an unfolded state below pH 4.5. The acid unfolding was extensively studied at pH 3.5 as a function of NaCl concentration. Addition of 0.05-0.2 M NaCl to a pH 3.5 sample increased the amount of beta-sheet structure with no change in the amount of alpha-helix and also induced reversible self-association of interferon gamma to form large aggregates from the monomer. When samples at pH 3.5 were dialyzed against 0.1 M ammonium acetate (pH 6.9) to refold interferon gamma, the samples that contained NaCl in acid formed aggregates upon dialysis while those without NaCl formed a dimer apparently identical with the starting protein (i.e., before acid treatment). Thus, the self-association of interferon gamma in acid is closely correlated with its aggregation behavior in 0.1 M ammonium acetate after removal of acid.

Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells.

Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.

Direct determination of macromolecular charge by equilibrium electrophoresis.

Charge is a fundamental property of macromolecules in solution. However, estimation of the apparent charge on polyions has confounded science for decades. Presented here is a general method to determine directly the apparent charge on a polyion, regardless of its size or shape. This new method uses equilibrium electrophoresis, a procedure in which opposing solute flows from electrophoresis and from diffusion balance everywhere as the system reaches a steady-state distribution. The method uses only small quantities of materials, is nondestructive, and requires only simple, inexpensive instrumentation. Here we describe a prototype apparatus, demonstrate the phenomenon, and present experimental examples of the procedure.

Structure of bovine blood coagulation factor Va. Determination of the subunit associations, molecular weights, and asymmetries by analytical ultracentrifugation.

Thrombin-activated bovine factor V (factor Va), an essential component of the blood clotting cascade pro thrombinase complex, is composed of two nonidentical subunits (Vl and Vh) and Ca2+ in tight association. We have examined Vl, Vh, and factor Va using analytical ultracentrifugation. At pH 7.65 in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 1 mM benzamidine, and 10 mM Ca2+, the Vl subunit has a molecular weight (Mr) of 82 500, an S0(20) ,w = 5. 0(2)S , and, assuming a model of a prolate ellipsoid with 0.3 g of H2O/g of protein, an axial ratio of 5:1. The corresponding values for the Vh subunit are an Mr of 92 300, an S0(20) ,w = 5.2(9) S, and an axial ratio of 5:1. We found these same values for Vl and for Vh in a buffer that contained 2 mM ethylenediaminetetraacetate (EDTA) rather than the 10 mM Ca2+. The Vl subunit undergoes a weak, reversible self-association at 9 degrees C with an apparent monomer-dimer association constant of 5.6 X 10(3) M-1 in the presence of 2 mM EDTA and 2.3 X 10(3) M-1 in the presence of 10 mM Ca2+. Our data indicate that the Vl self-association includes dimer and higher oligomers. Factor Va, examined in the presence of 10 mM Ca2+ and at 20 degrees C, has an Mr of 174 000, and S0(20) ,w = 8.1(8)S, an axial ratio of 5:1, and an apparent Vl-Vh association constant of at least 2.7 X 10(8) M-1. Our results suggest that factor Va self-associates to form higher multimers. When solutions of Va are dialyzed against a buffer that contains no Ca2+ and 2 mM EDTA, the apparent Vl-Vh subunit association constant is reduced to 9.4 X 10(3) M-1. Our hydrodynamic data indicate that there is a substantial decrease in molecular asymmetry when factor V is proteolytically activated by thrombin to form factor Va and that Vl and Vh are arranged "side by side" rather than "end to end" in factor Va.

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites.

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.

Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers.

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.

Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques.

Least-squares analysis of experimental data from the analytical ultracentrifuge is discussed in detail, with particular attention to the use of interference optics in studying nonideal self-associating macromolecular systems. Several samples are given that describe the application of the technique, the expected precision of the results, and some of its limitations. A FORTRAN IV computer program is available from the authors.