New revolutions in the evolution of analytical ultracentrifugation

650

New revolutions in the evolution of analytical ultracentrifugation Todd M Schuster* and John M Toedtt The use of the biophysical technique of analytical ultracentrifugation has recently undergone a resurgence. The commercial availability of the Beckman optima XL-A and XL-I analytical ultracentrifuges along with the continued growth in computing ability and analysis software has led to the expanded use of analytical ultracentrifugation and its capabilities. The genetic revolution and the search for further understanding of macromolecular interactions have again brought analytical ultracentrifugation to the forefront of macromolecular characterization.

Addresses Department of Molecular & Cell Biology and The National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut, 06269-3195, USA we-mail: [email protected] re-mail: JMt93001 @uconnvm.uconn.edu Current Opinion in Structural Biology 1996, 6:650-658

© Current Biology Ltd ISSN 0959-440X Abbreviations AU analyticalultracentrifugation RASMB reversible association in structural and molecular biology SCF stem cell factor S2o,w sedimentation coefficient corrected for 20"C and water

Introduction

It is now just over a decade since the introduction of recombinant DNA methodologies for the design and production of proteins. T h e success of these methods has contributed to establishing the ability to produce valuable commercial products and the capability of experimentally approaching the solutions to a number of fundamental questions in protein science, such as the mechanisms of protein folding and various aspects of the relationship between structure and function of proteins. Analytical uhracentrifugation (AU) was originally developed both theoretically and experimentally to become a leading method for the characterization of biological macromolecules. But in the 1960s the method began to lose popularity with biochemists because of the advances made in gel electrophoresis and gel chromatography. Furthermore, the AU instrumentation at that time lacked easily accessible automated data collection, and computer assisted data analysis was limited. There has been a vigorous rebirth of interest in AU in recent years, however, partly because of the commercial availability of newly designed instrumentation (e.g., Beckman XL-A and XL-I uhracentrifuges) which is highly automated for data collection and analysis. In addition, the developments of recombinant DNA

technology have created a renewed interest and need for a thermodynamically-sound, quantitative method for the analysis of protein solutions. As AU biophysical methods are so well suited for the quantitative characterization of the purity, size, shape, self-association and other binding properties of proteins in solution, it is rapidly becoming, once again, a necessary technique in most biochemical investigations. In addition, a number of advances have been made in the methods of computer-based data analysis and interpretation. AU methods are now orders of magnitude easier and faster to use, and more accurate than they were at the zenith of their popularity in the 1950s and 1960s. This is an outcome of the new, more sensitive and more stable instrumentation [1] and the widespread availability of high-speed, high-capacity laboratory computers which can be readily interfaced with the new or modified old analytical ultracentrifuges [2]. Accompanying the development of the improved instrumentation has been the availability of a plethora of new software programs that speed data acquisition, analysis, and interpretation. In the 1960s, the measurement of molecular weights and the characterization of association patterns for a simple protein that self associates could take several weeks, even in a well-equipped laboratory. Today such measurements can be done routinely in a few days [3]. Instead of being considered an out-of-date method, therefore, AU is now generally considered to be the method of choice for elucidating the following properties of biological (and other) macromolecules in solution [1,4]: size; size distribution; purity; gross conformation; thermodynamic nonideality (including virial and activity coefficients); equilibrium constants for self association, ligand binding and binding to other macromolecules; stability of macromolecular complexes; mechanisms of self assembly; and the characterization of the structure of gels and macromolecular network structures. In addition, although the AU is primarily a method for measuring macromolecules in relatively dilute solutions (concentration <10mgm1-1) it can also be used to study interactions in concentrated solutions [5], as well as in highly concentrated solutions such as gels, in order to determine thermodynamic and elastic properties [4]. T h e general versatility of AU is derived in part from the fact that it determines absolute thermodynamic parameters and does not depend on a calibration with macromolecules of known properties, in contrast to gel electrophoresis and gel chromatography [6]. Recently several review papers and monographs have appeared which document the status of the field and the wide range of its applications [3,7,8,9°',10]. Here we

Analytical ultracentrifugation Schuster and Toedt

attempt to expand on previous information and discuss the most recent developments in the field.

Online information One of the most modern and informative products of the renewed interest in AU is the on-line home pages and mail servers. Currently there are three notable sites that are extremely useful to both the novice and the expert in the field, and are excellent additions to the bookmarks of anyone interested in centrifugation. All of the pages have a complete bibliography of Beckman XL-A AU-related references in addition to their own unique features. The original site, RASMB (reversible association in structural and molecular biology), was created as a mail server to allow communication between workers in the field. It has grown over the past few years and now has a home page with a complete description of the file transfer protocl (ftp) site and list server, and instructions on their use [11"]. More than 100 scientists, including most of the pioneers and experts in the field, are members of the RASMB server and it offers an exceptional medium to discuss everything that is related to analysis software and instrumentation. The site also has an anonymous ftp site from which you can obtain programs for Mac, PC, and Dec/Vax computers. There are complete instructions on the home page of how to retrieve and deposit software. This feature has significantly assisted the expansion of the AU field. The software is now readily available to all users, and as all of the programmers are members of RASMB, the resources are available to all users, even to novices. A second, relatively new site which is hyperlinked via RASMB, is Bo Demelet's XL-A and RASMB site [12°°]. This site also has an extensive list of software that is downloadable for the analysis of equilibrium sedimentation and velocity sedimentation, and for data acquisition. In addition, this site has a searchable database of past RASMB communications and a complete e-mail list of current RASMB subscribers. Finally, this site has an outstanding tutorial on the van Holde-Weischet method, including data interpretation. This is an efficient way for those unfamiliar with the method to gain an understanding of the features of this velocity analysis software. The third site of interest is the Beckman home page [13"°], which, aside from containing information about their other commercial products, has an extensive list of background scientific papers and information about product development and data analysis for the XL-A and XL-I instruments. The exploration section has a complete description of techniques and explanations for studying solution interactions on the XL-A analytical ultracentrifuge. The references are written by experts in the field and are both a good initial source for the novice and a review source for the more experienced user.

651

Experimental approaches using AU There are two kinds of techniques, velocity and equilibrium sedimentation, that can be performed by AU to characterize macromolecules and their interactions in solutions. In a velocity-sedimentation experiment, the sample is exposed to a uniform, high-centrifugal field and the rate of movement of the particles that results is followed by measuring the sample concentration along the radius of the cell using either absorbance or interference optics inside the instrument. The rate of sedimentation of the resulting concentration boundary is governed by the magnitude of the centrifugal force applied and the amount of frictional force encountered by the sedimenting molecules (Fig. la). In addition, as the boundary is formed, diffusional flow also occurs. The diffusional force opposes the centrifugal force and results in a decreased flow and a spreading of the boundary with time. (Flow J =smZrc-D~ic/Sr). This factor can be seen in detail in the inset of Figure la where the spreading of the boundary is caused by a combination of diffusional force and the presence of multicomponents of the self-associating solute, tobacco mosaic virus coat protein. The results of a velocity-sedimentation experiment can be used to obtain information about the size, shape and purity of the sample, and about the occurrence of aggregation. Furthermore, in certain situations the molecular weight and diffusion coefficient (from which the molecular mass and shape of the molecule can be deduced) can also be determined. Equilibrium-sedimentation experiments are run at a lower rotor speed than a velocity sedimentation, which allows an equilibrium concentration gradient to form that can be analysed. The resulting boundary is a balance of the centrifugal force toward the bottom of the cell and the opposing diffusion force back across the boundary. The concentration boundary can be likened to a dialysis experiment whereby an imaginary membrane is formed by the centrifugal boundary, with the experimental solute trying to diffuse back across the imaginary membrane (Fig. lb). Equilibrium sedimentation is the method of choice for the determination of molecular weights and the characterization of association reactions. The analytical ultracentrifuge allows for a true thermodynamic analysis of equilibrium constants and a vast amount of information can be obtained about macromolecular-solution properties.

Velocity sedimentationdata analysis The classical method of analysis for velocity sedimentation is to follow the movement of the concentration boundary at the midpoint of the boundary. The results can then be plotted according to the Svedberg equation [14]: v S

-

-

o~2r

M(1 - 9 9)

Nf

652

Biophysical methods

Figure 1 (a) Velocity sedimentation experiment. Scans of tobacco mosaic virus (TMV) coat protein at 0.256 mg m1-1, 20" C, pH 70, 0.1 M ionic strength orthophosphate buffer, 60 000 rpm. Scans were recorded on a Beckman XL-A using absorption optics at 280 nm. Inset: Sequential scans taken at approximately 12 minute intervals (the left trace being the earliest scan). The earlier scans each show relatively sharp concentration boundaries compared with the later scans, which display shallower concentration gradients because of macro diffusion and partial resolution of the multiple aggregates of TMV coat protein. Each scan took approximately 2 minutes with settings of continuous mode, 0.005 cm step size, and a 5 point average (JM Toedt and TM Schuster, unpublished data). (b) Equilibrium sedimentation experiment. Single scan of TMV coat protein at 0.512 mg m1-1, 4"C, pH 7.0, 0.1 M ionic strength orthophosphate buffer, 32 000 rpm. The scan was recorded after 45 hours when equilibrium was confirmed by no further changes in the concentration boundary, on a Beckman XL-A using absorption optics at 280 nm (.IM Toedt and TM Schuster, unpublished data).

00

(a
08.

Meniscus

07" 060504

Centrifugational

03 020.1-

0.0 y ~ ' ~ -0.1-< -02 -03 -04 "0.5 "0.6 -07 -0.8 -0,9 -1.0 58

o8t

0.6 02

0.0 -0.2 "04

"06 -OB

-1.0

. . . . . . . . . . . . 5.8 5.9 6.0 61

6.2 6 3

6.4 6.5 6.6 6 7

59

60

5.1

62

63

6.4

65

66

57

6.5 6 9

7.0

(cm)

Radius

68

69

70

71

Radius(cm) 1 75

(b) 1.50

]

1.25 I

Centrifugational Force~t

1.00

0.75

~

Meniscus

0.50

0.25

// Diffusional ,~ Force

0.00

-0.25

"0.50 6.85

6.90

695

7.00

7.05

710

7 15

720

Radius (cm)

where s is the sedimentation coefficient in seconds, (oZr is the centrifugal force, to is angular velocity in revolutions per minute and r is the radial distance from the center of rotation), M is molecular weight, (1 - ~p) represents the buoyancy factor, f is the frictional coefficient, and v is the rate of sedimentation and can be expressed as dr/dt. Therefore, a plot of Ln rb(t)/rm(t0) versus (t-t0) will have a slope equal to to2s. This method can be used for simple systems, but as it does not account for diffusional spreading of the boundary or aggregational affects, more advanced methods of analysis are often required. There are several advanced analysis software packages available for velocity sedimentation (see the Online section), and each has unique features and advantages for specific

applications. It has been suggested by AU experts at the 1996 Annual National Analytical Uhracentrifugation Workshop at Storrs, Connecticut and at the workshop on advances in sedimentation velocity analysis at the 1996 Annual Meeting of the US Biophysical Society in Baltimore, MD, that all of the available programs should be used for analysis and so avoid misleading conclusions in situations where little knowledge is available for the system under study. T h e D C D T analysis program for velocity sedimentation data, developed by Walter Stafford, allows the calculation of the apparent s distribution, g(s*) [15]. T h e results of the analysis plotted as g(s*) versus s*, are nearly

Analytical ultracentrifugation Schuster and Toedt

653

It should be noted that this method does not correct for the effects of diffusion but one can extrapolate In g(s*) versus a quadratic in 1/t l/z to obtain the corrected distribution [17].

identical to the old schlieren optical system output (dc/dr versus r), and provide the ability to visually ascertain boundary information that can be difficult to obtain otherwise from the original scans (Fig. 2). It is easy, for instance, to determine the presence of multiple components or aggregates. T h e major advantage of this method is the increase in signal-to-noise ratio as a consequence of the combination of differentiation with respect to time (which results in a complete elimination of time-independent baseline elements) and the averaging of the time-derivative patterns. These procedures result in an increase of about 2-3 orders of magnitude in the sensitivity and allow analysis of solutions of much lower concentration [16]. It is now possible to investigate boundaries with concentrations <101aml-1 when they are obtained with a rapid acquisition video-based Rayleigh optical system [2].

A new innovation involving the use of this method is the ability to determine the molecular weight of an ideal monodispersed macromolecule from the standard deviation of the g(s*) versus s* plot [18]. It has been shown that the diffusion coefficient can be related to the standard deviation of the resulting Gaussian curve obtained from the D C D T program by the relationship: D = ((~rmm2t)z/zt where o is the standard deviation of the g(s*) curve. Therefore, via the Svedberg equation, the molecular weight can be calculated. T h e method offers a

Figure 2 Velocity sedimentation analysis using DCDT analysis [15]. (a) The results of the first step of the time derivative analysis are shown. The plot of dc/dt versus s is shown for scan numbers 30 to 41 of the velocity experiment described in Figure la. (b) The results of the second step of the time derivative method are shown (< g ^ (s*) > versus s). It can be seen by the non-Gaussian shape that there are multiple components that make up the sedimentating boundary. The weight average (< S > w) was calculated to be 2.89_+0.22 S for the entire boundary and the weight-average sedimentation coefficient corrected for 20"and for water (< S2o,w> w) was calculated at 2.98 S. Inset: The < g ^ (s*)> versus S plot is shown again with the resulting error bars (based on the original unsmoothed data) which confirm that the smoothing of the data did not distort the output.

(a)

ae-S

•~

,

o°+Ooo

0.5

10

IS

20

25

3.0

3.5

40

45

51o

si~

,o

s

o,. i (b) o16-~w= 2 . 9 8

0.12

o,s S

J

~

/ o

~

0.00

\ i

/

~

,

654

Biophysicalmethods

quick way to determine the molecular weight of simple macromolecules, with an uncertainty of about 10%. A second method available for the analysis of sedimentation velocity experiments (see the Online section) is the van Holde-Weischet method [19], which calculates an integral distribution of s. T h e method first divides each AU scan into n divisions and the apparent sedimentation coefficient is determined for each point, n. These values are then plotted versus 1/tl/2 and the data (s*) from each scan of the n points are extrapolated to infinite time (Fig. 3a). The y-intercept is equal to the diffusion-corrected sedimentation coefficient. T h e average of the y intercepts is used to calculate a weight-average sedimentation coefficient, Sw. T h e results are then plotted as fraction of total sedimenting material versus s (Fig. 3b) and allow the user to make several important visual interpretations about the macromolecule being investigated [3,12°°]. A homogenous solution will result in a single y-intercept in the s versus 1/tl/2 plot and a vertical line for the fraction versus s plot. Figures 3a,b display the output for the same experiment analyzed in Figure 2. T h e results depict a self-associating system, as deduced from the multiple y-intercepts in Figure 3a and a slope in Figure B which arise from the multiple s values obtained. These plots allow investigators to make several conclusions about the system in question, and have a number of possible applications for studies involving the characterization of macromolecules, for example in sample quality control. A third type of analysis available is the method by Holladay [20,21] and Philo [22] (see Online) that fit velocity data to models derived from approximations of the L a m m equation. The advantage of this method is that it results in the determination of both s and D (the diffusion coefficient). Therefore, one can easily calculate the molecular weight and additional hydrodynamic parameters. T h e Philo method also has the ability to analyze for up to three noninteracting components if their s values differ by a factor of about 1.5 or greater. It should be stressed, however, that the fit is not appropriate for interacting systems and other methods should be used in cases of interacting macromolecules [22].

dln(c) dr2~2

mZs

D

M2(1 - 9 9 ) 0 2 RT

This equation provides the theoretical basis of analyzing sedimentation equilibrium data [24]. A plot of In(c) versus r2/2 for a monodisperse macromolecular system will be linear and will give a slope, M2(1 -gp)oa2/RT, that provides the weight-average molecular weight of the solute.

For a polydisperse system, the above plot will curve upward and the tangent of the curve gives the weight-average molecular weight using the appropriate manipulation. One may also compute the so-called number and Z-average molecular weights as a function of concentration and radial position. T h e analysis of equilibrium sedimentation data is best accomplished through the use of computer programs that fit the data for various models such as ideal, nonideal, and assorted self-associations. T h e N O N L I N program [25] and the Omega [26] analysis are two programs designed for detailed analysis of this type. For a detailed discussion of the analysis of sedimentation equilibrium data, see Johnson and Straume [27] and references therein. There is a vast amount of literature citing the use of velocity sedimentation and equilibrium sedimentation techniques in a variety of applications [28°,29-32,33",34",35-37]. T h e reference lists available online are an excellent source to find documents about specific systems. In particular, the Beckman reference list [13 "°] has a breakdown of XL-A and XL-I use on the basis of the type of biological system investigated. In this section, we will present two examples of how AU has been used recently. A superb example of one use of AU is illustrated by the physical characterization of calponin [38°]. T h e study characterized the protein using the combined techniques of circular dichroism, electron microscopy, and AU sedimentation equilibrium and velocity analysis. Calponin is a thin filament-associated smooth muscle protein that has been suggested to play a role in the regulation of smooth muscle contraction. T h e aim of the study was to gain a better understanding of how calponin interacts with actin and of possible interactions with other actin-associated proteins.

Equilibrium sedimentation data analysis

Sedimentation equilibrium can be used to measure molecular weights over a wide range of sizes, from small molecules such as sucrose (360 gmo1-1) to large viruses like tobacco mosaic virus (40 x 106 gmol-l). At sedimentation equilibrium, the net transport between sedimentation and diffusion is zero, and there are no further changes in the concentration profile across the sample cell with time. Using the Lamm equation for hydrodynamic flow combined with the Svedberg equation [7,23] it can be shown that:

AU was used to first determine calponin's molecular weight and its degree of association using equilibrium sedimentation. T h e resulting concentration gradients, obtained by a Beckman model E equipped with a real-time video-based data acquisition system and Rayleigh optics [2,39], were analyzed using the program N O N L I N [25]. T h e results revealed a monomer, molecular mass of 31.4+l.0kDa, which is in excellent agreement with the value of 32.3kDa calculated from the amino acid composition. Furthermore, the results showed that at

Analytical ultracentrifugation Schuster and Toedt

655

Figure 3 Velocity sedimentation analysis using the van Holde-Weischet method. (a) The resulting van Holde-Weischet plot for the TMV coat protein data described in Figure la. The resulting y intercept represents the diffusion-corrected S2o,w value. The multiple y intercepts are the result of multiple components that make up the TMV coat protein sedimentation boundary. The resulting < S20,w > w value is 2.93 S. (b) Second plot of the van Holde-Weischet analysis, cumulative fraction versus S2o,w. The output displays various fractions with differing S values, again because of the multiple components of the self-associating TMV coat protein.

5.0-

4.54.0o~. 3.5-

2,5-

2.01.51.00.000

0.005

0.015

0.010

0.020

0.025

0.030

T i m e (-0.5) I

1.0-

I

I

I

(b)

O e e o

0.8 o o o e

¢.o

0.6 e e e

i1

'5 0.4 E O

o o e

0.2

0.0 2.75

I

I

I

I

2.80

2.85

2.90

2.95

3.00

S20,w

concentrations of 1.2mgml-1 in a buffer containing 0.1 M NaCI the protein was monodispersed. Velocity sedimentation scans collected with the on-line Rayleigh system were analyzed using the D C D T program. The analysis yielded an S20,w value of 2.34S. This sedimentation coefficient was then used to determine the size and shape of the protein. Using a reasonable value for the hydration of a protein and a partial specific volume estimated from the amino acid composition, the f/fo ratio was calculated. The ratio was then used to model the calponin protein as a prolate ellipsoid, yielding an

axial ratio of 6.16, a length of 16.2nm and a diameter of 2.6nm, which is in excellent agreement with the length obtained from electron microscopy. The length evaluation was then used to model a possible binding stoichiometry of actin:calponin. The results of the study revealed that calponin is a flexible elongated molecule with a contour length of 16.2nm, which would allow for a 3:1 stoichiometric relationship of actin:calponin. Interestingly, this investigation also used AU as a tool to determine the extinction coefficient of a protein. An extinction coefficient of 0.74(mgml-l)-lcm-1 was obtained for by using the Rayleigh interferometric optical

656

Biophysicalmethods

system of the AU to measure the refractive index of the protein solution and hence protein concentration. This capability will be easily exploited in the Beckman XL-I, which has both interference and absorption optics, and a highly automated data collection system. T h e second interesting AU investigation, carried out very recently by Philo et al. [40], was on the interactions between the human stem cell factor (SCF) and its receptor, the tyrosine kinase, Kit. Sedimentation equilibrium analysis was used to investigate this A + B interaction and obtain information on its possible in vitro stoichiometry and association constants. The ability to research such complex interactions with AU is because of the improved data acquisition, data analysis software, and computer advancements. An investigation of the interactions between two different macromolecules requires a comprehensive understanding of the properties of the individual macromolecules. This requirement demands that a full characterization of the macromolecular properties must be determined before attempting to obtain interaction

information about the A + B complex. Therefore, both Kit and SCF were examined individually by sedimentation equilibrium techniques using the Beckman XL-A analytical ultracentrifuge. T h e results of the equilibrium analysis revealed that SCF is dimeric whereas Kit behaves as an ideal single monomeric species under the conditions of the study. Size exclusion chromatography was first used to study the stoichiometry of the Kit-SCF complex by the analysis of various molar ratios of the two components. T h e results strongly indicated the formation of a complex containing equimolar amounts of Kit and SCF and that Kit-SCF could therefore exist as a 1:1 or a 2:2 complex. To distinguish between the two possibilities, size exclusion chromatography was repeated, in conjunction with light scattering detection, to determine the molecular weights of the eluting species. T h e molecular weights indicated that the complex contained two Kit and two SCF monomers (one SCF dimer). Sedimentation equilibrium analysis was also employed to examine the complexes and the results clearly confirmed that the sample contained the 2:2 complex. In addition, sedimentation equilibrium analysis

Figure 4

I

Defining the Interactions of Macromolecules in Solution:

1

I

Mas0eotroet1 I Resonance P I I Anaca

Ultracentrifugation

Molecular

Mass

Non-covalent

Interactions, ~

Stoichiometry~

Covalent Modifications

Macromolecular Assay Design 1 Molar Ratio 1

Identification data bases

IcaTitorrialmiOr~ry)

Enzyme Assay

L Fluor., CD, LSJ

I Differential Scanning | Calorimetry)

Enzyme Assay, Fast Kinetics

Mass ~

k a 101- 106 M-t s-t kd 10-2-10"6 s"1

of unknowns from

Solution Molecular

Binding Kinetics

Epitope Mapping

]

I r Spectroscopyl

Assembly

States, Stoichiometry~shape

~

/ Conformational

Changes

Thermodynamics, Free Energy

Molar Ratio ~

Thermodynamics, Free Energy and Enthalpy

Molar Ratio

Thermal Stability l

Conformational Changes

Conformational Changes

Thermal Stability

Affinity, Kd

Affinity, Kd

Affinity,

Affinity,

Affinity,

10-4-10-11 M

10-3-10-8 M

10-6-10-11M

10-6-10-11 M

10-9-10-2o M

Kd

Kd

K~

A schematic representation of the relationships between various biophysical methods used in the characterization of biological macromolecules. The range of binding constantsthat are detectableby each method is shown with an overview of how the various methods complement each other. Printed with permission from P Hensley.

Analytical ultracentrifugation Schuster and Toedt

was utilized to fit a model derived from the preliminary data to obtain a dissociation constant. The data obtained for samples run at three different speeds and five different loading concentrations were globally fit to a model as described in the text [40] in which each SCF direct has two equivalent sites to which the Kit molecules bind independently. The best fit using all 2548 data points gave a dissociation constant of 17 nM.

Conclusion The past five years have seen an explosive rebirth of interest in AU. Some of the reasons for this renaissance derive from the commercial need to quantitatively evaluate and characterize pharmaceutical formulations of new protein products that are being developed using modern recombinant DNA and cell-culture methods. Such investigations are being supported by increasingly sophisticated, yet accessible instruments and computer programs for data acquisition, analysis and interpretation. Whereas other methods are better suited for a rapid survey of the qualitative aspects of ligand binding and other macromolecular interactions, AU is probably the best method for obtaining quantitative information on the thermodynamic characterization of such interactions. AU has a relatively large number of users world-wide. There are now several sources of scientific information and support available to the wider scientific community through workshops, publications, scientific conferences and rapid communication via the Internet. In addition, direct communications are available with National Resource Facilities in the United States [41] and United Kingdom [42], as well as with other research laboratories with individuals highly experienced in the methodology of AU. Several recent scientific conferences and workshops have revealed that this is a community of AU researchers who not only regularly communicate with each other via the Internet, but who also willingly enter into research collaborations with colleagues who have limited experience with AU practice. There are ongoing efforts to improve ultracentrifuge equipment and advance the acquisition and analysis of more precise data by using the latest computational methods as they become commercially available. One can expect a continued strong interest in AU as the field of molecular biotechnology develops further. Alongside this, there will be an need for trained laboratory personnel who can successfully implement the wide range of developments in AU methodology currently available and currently being pursued. As research interests turn to more complex problems, such as the mechanisms of macromolecular assembly, it will be necessary to employ a wide range of biophysical methodologies and integrate the results, in order to obtain a clear and accurate picture of mechanistic details, including quantitation of the energetics of the relevant binding processes such as

657

the thermodynamic parameters of the reaction enthalpies and entropies as well as intermolecular free energies. For example, AU currently bridges the range of binding constants that can be determined by two other popular biophysical methods, as is shown in Figure 4, which schematically relates the ranges of applicability of AU in determining thermodynamic binding constants to the ranges for two other methods. Of course, the exact range that can be covered by each method depends upon intrinsic properties of the system under study, such as extinction coefficient, enthalpy of reaction, nonspecific binding etc. Figure 4 reminds us that it is desirable to combine results from several quantitative methods to develop a detailed molecular mechanism for a macromolecular-assembly process. Such detailed mechanisms may then be interpreted in terms of atomic-resolution structural data as obtained from X-ray crystallograph3; NMR and mass spectrometry.

Acknowledgements This work was supported by the Division of Biological Instrumentation and Resources, US National Science Foundation, Arlington, VA, USA. \\'e thank Preston Hensley for the preparation of Figure 4.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • o.

of special interest of outstanding interest

1.

Laue TM: On-line data acquisition and analysis from the rayleigh interferometer. In Analytical U/tracentrifugation in Biochemistry and Polymer Science. Edited by Harding SE, Rowe AJ. Cambridge, UK: Royal Society of Chemistry; 1992.

2.

YphantisDA, Stafford WF, Liu S, Olsen PH, Lary JW, Hayes DB, Moody TP, Ridgeway TM, Lyons DA, Laue TM: Online data acquisition for the Rayleigh interference optical system of the analytical ultracentrifuge, In Modern Analytical U/tracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:209-226.

3.

HansenJC, Lebowitz J, Demeler B: Analytical ultracentrifugation of complex macromolecular systems. Biochemistry 1994, 33:13155-131 63.

4.

Harding SE: The sedimentation equilibrium analysis polysaccharides and mucins: a guided tour of the problem solving for difficult heterogeneous systems. In Modem Analytical Ultracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:315-342.

5.

Minton AP: Conservation of signal: a new algorithm for the elimination of the reference concentration as an independently variable parameter in the analysis of sedimentation equilibrium. In Modern Analytical U/tracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:81-93.

6.

Fujita H: Notes on the derivation of sedimentation equilibrium equations. In Modern Analytical U/tracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:3-15.

7

Schuster TM, Laue TM (Eds): Modern Analytical Ultracentrifugation. Boston: Birkhauser; 1994.

8.

Harding SE: The analytical ultracentrifuge spins again. Trends

Anal Chem 1994, 13:439-446. 9. •-

Hensley P: Defining the structure and stability of macromolecular assemblies in solution: the re-emergence of analytical ultracentrifugation as a practical tool. Structure 1996, 4:367-373. Presents the use of AU in conjunction with other biophysical methods in the characterization of macromolecules and macromolecular interactions. Mass spectrometry, surface plasmon resonance, isothermal titration calorimetry, differential scanning calorimetry, and spectral approaches are examined to

distinguish their uses both alone and in combination with each other and AU. In addition, sedimentation velocity analysis, (in particular the DCDT method) and equilibrium sedimentation analysis are reviewed. 10.

Harding SE: Analytical ultracentrifugation and the genetic engineering of macromotecules. Biotechnol Genet Eng Rev 1993, 11:317-356.

11. RASMB Home Page on World Wide Web URL: http://bbri;o www.eri.harvard.edu/RASMB/rasmb.html r contents of the home page, see text. The available software includes that from Stafford, Johnson and Yphantis/National Analytical Ultracentrifuge Facility, Minton, Holladay, Philo, Demeler, and Ralston. Information about the mail server is also given. You can join the server by e-mailing [email protected].

Ultracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:37-65. 28. •

Advant S J, Braswell EH, Kumar CV, Kalonia DS: The effect of pH and temperature on the self-association of recombinant human interleukin-2 as studied by equilibrium sedimentation. Pharm Res 1995, 12:637-641. The self-association of recombinant human interleukin-2 in solution was investigated as a function of pH and temperature using equilibrium sedimentation at 10"C and 20°C. The data at both both temperatures are best described by a weak monomer-dimer association equilibrium. 29.

Henniker A, Ralston GB: Reinvestigation of the thermodynamics of spectrin self-association. Biophys Chem 1994, 52:251-258.

Bo Demeler's XL-A and RASMB/Analytical Ultracentrifugation page at the UTHSCSA Biochemistry Department on World Wide Web URL:http://biocO2.uthscsa.edu/.biochem/x/a.htm/ This site has the same downloadable software available on the RASMB site [11"'] but in addition it has Jeff Hansen's AU review article, the RASMB mailing list with past communications, collaboration and fee-for-service information, a tutorial on the van Holde-Weischet method, and data analysis examples.

30.

Behal RH, DeBuysere MS, Demeler B, Hansen JC, Olson MS: Pyruvate dehydrogenase multienzyme complex: characterization of assemble intermediates by sedimentation velocity analysis. J Bio/Chem 1994, 296:31372-31377

31.

Darawshe S, Minton AP: Quantitative characterization of macromolecular associations in solution via real-time and postcentrifugation measurements of sedimentation equilibrium (SE): a comparison. Ana/Biochem 1994, 220:1-4.

13. Beckman Home Page on World Wide Web URL: •. http://www.beckman.com This is the main Beckman home page. From here you can easily access the Optima XL-A application and reference list, the XL-A/XL-I description at and the exploration site. The reference site here is exceptional because the XL-A applications are separated into several categories which include binding studies and complexes, general studies, glycoproteins and proteoglycans, lipoproteins, miscellaneous samples (including peptides), new, nucleic acids, proteins (enzymes), proteins (receptors), other proteins, synthetic polymers, and viruses.

32.

Zarina S, Slingsby C, Jaenicke R, Zaldi ZH, Driessen H, Srinivasan N: Three-dimensional model and quaternary structure of the human eye lens protein yS-crystallin based on ]3- and y-crystallin X-ray coordinaltes and ultracentrifugation. Protein Sci 1994, 3:184-186.

12. ••

14.

Cantor CR, Schimmel PR: Biophysical Chemistry, Part II. San Francisco: W.H. Freeman and Company; 1980.

15.

Stafford WF II1: Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal Biochem 1992, 203:295-301

16.

Stafford WF IIh Sedimentation boundary analysis of interacting systems: use of the apparent sedimentation coefficient distribution function. In Modern Analytical Ultracentrilugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:119-137.

17.

Stafford WF II1: Boundary analysis in sedimentation velocity experiments. Methods Enzymol 1994, 240:478-501.

18.

Stafford WF II1: Rapid molecular weight determination by sedimentation velocity analysis [abstract]. Biophys J 1996, 70:A231.

19.

Van Holde KE, Weischet W: Boundary analysis of sedimentation-velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 1978, 17:1387-1403.

20.

Holladay LA: An approximate solution to the Lamm equation. Biophys Chem 19?9, 10:187-190.

21.

22.

23.

33. •

Wu Z, Johnson KW, Goldstein B, Choi Y, Eaton SF, Laue TM, Ciardelli TL: Solution assembly of a soluble, heteromeric, high affinity interleukin-2 receptor complex. E Bio/Chem 1995, 270:16039-16044. In this biophysical study, the authors report the use of coiled-coil (leucine zipper) molecular recognition for the solution assembly of stable, high affinity, heteromeric interleukin-2 receptor complexes. Interleukin-2 receptor and extracellular domains, each fused to seven coiled-coil heptad repeats, resuited in the formation of heteromeric complexes that bound interleukin-2 in a cooperative fashion and with much higher affinity than similar homomeric complexes. 34. •

Correia JJ, Gilbert SP, Moyer ML, Johnson KA: Sedimentation studies on the kinesin motor domain constructs K401, K366 and K341. Biochemistry 1995, 34:4898-4907. AU studies of bacterially expressed kinesin motor domains revealed that domain K401 is predominantly a dimer in solution but is able to associate into higher oligomers by a 1 - 2 - 4 mechanism. Values of the dissociation constant in the presence and absence of ATP were obtained. A good discussion of experimental procedure is presented. 35.

Hensley P, McDevitt PJ, Brooks I, Trill JJ, Field JA, McNulty DE, Connor JR, Griswold DE, Kumar NV, Kopple KD etaL: THe soluble form of E-selectin is an asymmetric monomer: expression, purification, and characterizion of the recombinant protein. J Biol Chem 1994, 269:23949-23958.

36.

Venugopal MG, Ramshaw JAM, Braswell E, Zhu D, Brodsky B: Electrostatic interactions in collagen-like triple-helical peptides. Biochemistry 1994, 33:7948-7956.

Holladay LA: Simultaneous rapid estimation of sedimentation coefficient and molecular weight. Biophys Chem 1980, 11:303-308.

37.

Philo JS: Measuring sedimentation, diffusion, and molecular weights of small molecules by direct fitting of sedimentation velocity concentration profiles. In Modern Analytical Ultracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:156-170.

Kim S, Tsukiyama T, Lewis MS, Wu C: Interaction of the DNAbinding domain of Drosophila heat shock factor with its cognate DNA site: a thermodynamic analysis using analytical ultracentrifugation. Protein Sci 1994, 3:1040-1050.

38. •

Stafford WF, Schuster TM: Hydrodynamic methods, In Introduction to Biophysical Methods for Protein and Nucleic Acid Research. Edited by Glasel JA, Deutscher ME New York: Academic Press; 1995:111-143.

24.

Johnson ML, Frasier SG: Nonlinear least-squares analysis. Methods Enzymol 1985, 117:301-341.

25.

Johnson ML, Correia JJ, Yphantis DA, Halvorson HR: Analysis of data from the analytical ultracentrifuge by nonlinear leastsquares techniques. Biophys J 1981, 36:575-585.

26.

Winzor D J, Wills PR: Omega analysis and the characterization of solute self-Association by sedimentation equilibrium. In Modern Analytical Ultracentrifugation. Edited by Schuster TM, Laue TM. Boston: Birkhauser; 1994:66-80.

27.

Johnson ML, Straume M: Comments on the analysis of sedimentation equilibrium experiments. In Modern Analytical

Stafford WE Mabuchi K, Takahashi K, Toa T: Physical characterization of calponin. J Biol Chem 1995, 270:10576-10579. This reference is discussed in detail in the text. It is a classic example of how AU can be used to determine size and shape of a macromolecule. 39.

Stafford WF III, Liu S: A real-time video-based Rayleigh optical system for an analytical ultracentrifuge allowing imaging of the entire centrifuge cell [abstract]. Biophys J 1992, 61 :A476

40.

Philo JS, Wen J, Wypych J, Schwartz MG, Mendiaz E, Langley K: Human stem cell factor dimer forms a complex with two molecules of the extracellular domain of its receptor, Kit. J Biol Chem 1996, 271:6895-6902.

41.

The National Analytical Ultracentrifugation Facility at the University of Connecticut on World Wide Web URL: http://gopher.uconn.edu:80/-wwwbiotc/ua(html

42.

The UK National Centre for Macromolecular Hydrodynamics (NCMH) on World Wide Web URL: http://www.ccc.nottingham.ac.uk/-sczles/ncmhpage.html