NASA Technical Memorandum 107120

A Fiber Optic Probe for Monitoring

Protein Aggregation, Nucleation,

and Crystallization

Rafat R. Ansari and Kwang I. Suh

Lewis Research Center

Cleveland, Ohio

Alireza Arabshahi and W. William Wilson

Mississippi State University

Mississippi State University, Mississippi

Terry L. Bray and Lawrence J. DeLucas

University of Alabama at Birmingham

Birmingham, Alabama

Prepared for the

Sixth International Conference on Crystallization of Biological Macromolecules

sponsored by the Hiroshima Prefecture, Hiroshima City, the Ministry of Education,

Science, Sports and Culture, and the International Union of Crystallography

Hiroshima, Japan, November 12–17, 1995

National Aeronautics and

Space Administration
NASA TM–107120 1

A FIBER OPTIC PROBE FOR MONITORING PROTEIN AGGREGATION,

NUCLEATION, AND CRYSTALLIZATION

R.R. Ansari and K.I. Suh

National Aeronautics and Space Administration

Lewis Research Center

Cleveland, Ohio 44135

A. Arabshahi and W.W. Wilson

Mississippi State University

Mississippi State, Mississippi 39762

and

T.L. Bray and L.J. DeLucas

The University of Alabama

Birmingham, Alabama 35294

SUMMARY

Protein crystals are often experimentally grown in hanging drops in microgravity experiments on-board the

Space Shuttle orbiter. The technique of dynamic light scattering (DLS) can be used to monitor crystal growth processes in hanging droplets (~30 μL) in microgravity experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we demonstrate that such experiments are now

feasible. We apply a newly developed fiber optic probe to various earth and space (microgravity) protein crystallization system configurations to test its capabilities. These include conventional batch (cuvette or capillary) systems,

a hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for

temperature-induced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within

a few seconds) means of quantitatively and noninvasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for performing single-angle static light scattering

measurements. It utilizes extremely low levels of laser power ( a few μW) and essentially eliminates the usual problems associated with optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for visualization of macroscopic protein crystals. This new optical diagnostic system makes

possible the exploration of new ways to grow good quality crystals suitable for x-ray crystallographic analysis and

may contribute to a concrete scientific basis for understanding the process of crystallization.

INTRODUCTION

The growth of protein crystals is usually the rate limiting step in the determination of three dimensional structures of biologically important proteins. This area has been of particular interest in recent years due to the use of

structure based rational drug design for developing new pharmaceutical materials. This approach relies on the determination of the three dimensional structure of a specific protein related to a particular disease process. Once the

structure has been determined and the appropriate active site(s) of the protein have been identified, then a drug can

be designed to modify the protein's activity and interrupt the disease process. The only direct method for determining three dimensional structures of large biological proteins is the technique of x-ray crystallography which requires

well ordered crystals of the target protein in order to solve the structure. Obtaining these crystals is typically the

most difficult step prior to using x-ray crystallography. This difficulty led to the exploration of other avenues which

might yield high quality crystals. Early experiments by Walter Littke (ref. 1) led researchers in the U.S. to investigate the effects of eliminating gravity on crystal growth. This interest ultimately resulted in the development of a

hanging drop vapor diffusion apparatus (HDVDA). This hardware, described by Rosenblum et al. (ref. 2), has been
 2 NASA TM–107120

used on Space Shuttle missions to successfully grow crystals of several proteins which have produced x-ray data

surpassing the data collected from the best earth grown crystals. However, not all proteins have produced better

crystals in space. This is believed to occur because the conditions for crystal growth are different in a microgravity

environment than in 1g.

The fundamental physics for crystallization of biological macromolecules is still not well understood. Small

molecules (e.g., NaCl) have stronger bonds holding the crystal together than do protein crystals which are typically

held together by much weaker electrostatic interactions, hydrogen bonds, and water bridges. Protein crystals also

contain a great deal of solvent, typically anywhere from 40 to 80 percent of the crystal volume. These factors make

protein crystals much more difficult to crystallize in a well ordered manner suitable for x-ray diffraction studies.

While there are several theories regarding the mechanism of protein crystallization, it is not definitively clear

whether monomers or some higher order aggregate(s) comprise the growth unit. There is evidence supporting both

theories. It is possible both mechanisms contribute to crystal growth simultaneously. A concrete physical understanding of the protein crystallization process at a fundamental science level would require detailed studies of the

nucleation and post-nucleation growth phenomena. Since nucleation may depend upon temperature, monomer concentration, pH, solution ionic strength and crystallizing agent concentration, dynamic control of one or more of these

parameters and a specific monitoring technique would be very useful. Dynamic control of nucleation offers the possibility of a more systematic approach to protein crystal growth, and its implementation calls for the development

of suitable quantitative and noninvasive monitoring techniques.

Since the evaporation rate in a HDVDA experiment is set by the initial solution concentrations, active control

over the equilibration rate of the growth solution is not possible once the experiment is activated. This limitation

has led to the development of a dynamically controlled vapor diffusion system (DCVDS), described by Bray et al.

(ref. 3), which does allow active control over evaporation rates. The DCVDS utilizes humidity sensors and a laser

scattering detector which allow control over the approach to nucleation, detection of nucleation and control of postnucleation growth. The major goal of this study is to determine if the optical probe described in this paper provides

the necessary features as a laser scattering diagnostic so that it meets the requirements for the DCVDS and other

ground based and space based systems. The preliminary results indicate that the probe does meet this objective.

Data Analysis

The technique of DLS is commonly used in the characterization of macromolecular solutions and its principles

are documented in several books such as the one by Chu (ref. 4). The incident laser radiation interacts with the particles dispersed in a fluid. The particles scatter light and the intensity of this scattered light fluctuates in time due to

the Brownian or thermal motion of the dispersed particles in the suspending medium. When a dynamic medium such

as a protein solution is illuminated by laser light, a rapidly changing interference pattern due to the differences in the

optical density of the scattering medium is produced and contains information about the dynamics of the scattering

medium which can be extracted by constructing a time autocorrelation function (TCF) (refs. 4 and 5). The TCF is

computed using a digital correlator, and in the case of dilute dispersions of spherical particles, the TCF provides

quick and accurate determination of the particle translation diffusion coefficient. The diffusion coefficient data can

easily be transformed into average particle size, using the Stokes-Einstein equation, provided the viscosity of the

suspending medium, its temperature and refractive index are known.

Conventionally the technique of DLS has been used for studying dilute solution properties of spherically shaped

particles of effective size ranging between 3 nm and 3 μm. For concentrated suspensions exhibiting multiple light

scattering effects and for dispersions containing more than one scattering species, however, the data analysis and

interpretation of the TCF becomes more difficult. The protein systems studied in this paper do not exhibit multiple

light scattering effects. Our DLS probe is capable of handling concentrated dispersions. Ansari et al. (ref. 6) have

accurately determined, without a need to correct for multiple light scattering, the alpha crystalline protein size in

the eye lens where its composition is very high (35 to 40 percent w/ν). We must note, however, that the TCF data

obtained during post-nucleation protein crystal growth where nonspherical micron sized particles are present must

be analyzed and interpreted with extreme care. When particle size approaches ~1 μm in ground-based systems, sedimentation often becomes dominant. Therefore, DLS data should only be used to detect gross trends when particle

sizes become larger and the assignment of specific size values in absolute terms is qualitative. The TCF data presented here was analyzed using the commercial data inversion routines supplied by the Brookhaven Instruments
NASA TM–107120 3

Corporation (Holtsville, NY). These include a method of cumulants, double exponential, and a computer algorithm

called CONTIN. These data analysis techniques and their comparison are described in detail by Stock and Ray

(ref. 7).

Optical Design

We chose to exploit the technique of DLS to reliably characterize dynamical properties (diffusion coefficients

or size) of the protein molecules in a hanging drop. Until recently such experiments were not possible because of

optical alignment problems, control of penetration depth inside the sample, physical size of the instrumentation and

power requirements for the laser and photodetectors. In the last few years, however, the DLS instrumentation has

profited from technologies associated with the advent of miniaturization in solid state lasers, photodetectors, optical

components and fiber optic communication devices (ref. 8).

One of the difficulties in probing protein monomers in dilute solutions is their small size (~3 to 8 nm in diam).

Conventional DLS systems require laser powers of the order of a few hundred milliwatts and data collection times

of minutes to obtain a good-quality TCF. One factor affecting the TCF, as shown by Ford (ref. 5), is the precise control of the scattering volume or coherence area in the sample. In conventional DLS systems the coherence area is

controlled by making use of lenses and apertures as part of the launch and detection optics. Fiber optic systems reported by Dhadwal (ref. 9) have made use of two optical fibers, one for launch and the other for detection, mounted

on one gradient index (GRIN) microlens. A lensless fiber optic system for doing DLS measurements has also been

reported by Dhadwal et al. (ref. 10). The former (fig. 1 and 1(a) in ref. 9) can only be used as an immersion probe

while the lensless probe (ref. 10) can be used both inside and outside the sample cell. Both designs have certain limitations. In the former case, in our experience, reflections arising from the lens and fiber surfaces give rise to heterodyning effects thus compromising the reliability and accuracy in obtaining correct particle sizes. The lensless fiber

optic system, on the other hand, solves the problem of spurious reflection, but for outside use it must be placed

within a very close proximity (< 2 mm) of the sample being interrogated. Further, its use is limited in interrogating

very dilute samples when used in conjunction with solid-state lasers and samples of larger colloidal particles. The

sensitivity and reliability are drastically reduced for particles greater than 300 to 500 nm because of extremely low

spatial coherence (β) values and a large scattering area (~5 mm2). This system is therefore not suitable for the

HDVDA or DCVDS system configurations.

Casay and Wilson (ref. 11) have successfully demonstrated the use of static and DLS techniques in the only

reported study of the lysozyme nucleation process in a modified HDVDA. In that study separate laser launch and

detection systems were used. The laser launch system employed a multimode optical fiber with a microlens to produce a collimated beam of light in the sample. The scattered light was collected at a scattering angle of 90° using a

single mode optical fiber. The separate laser and detector systems compromise flexibility and demand a high degree

of optical alignment which is not easy to achieve in a microgravity environment. Although the experimental results

were good, their system was not optimized for the most efficient self-beating (DLS) light scattering experiments.

Further, such a system cannot be used on a DCVDS system which employs sample cells fabricated out of acrylic

material which becomes opaque upon machining.

Our newly designed system alleviates all of the above problems. We attach separate micro-graded index

(GRIN) lenses on each optical fiber (launch and detection) in such a way that the scattering volume (~2×10–5 mm3)

and coherence area (ref. 5) are very tightly controlled resulting in high spatial coherence (β >0.5) values. A fiber

optic probe comprised of two monomode optical fibers and two GRIN microlenses, as illustrated in figure 1, provides a compact and remote means of studying the dynamical characteristics of the protein particles in a hanging

droplet. The fiber optic probe (1.3 cm in diam × 1.3 cm in length) contains the necessary optics to perform DLS

measurements at a scattering angle of 161.5°. The probe is shown in figure 1 with a U.S. dime for size comparison.

The probe is noninvasive and is conveniently positioned in front of the drop, but has no physical contact with any

part of the sample or its container. Two monomode optical fibers, each housed in a stainless steel ferrule, are

mounted into a separate stainless steel housing. An air gap ( 0 to 0.05 mm) is intentionally left between the fiber

housing and the lens housing in order to produce a tightly focused spot in the scattering volume. The two optical

fibers in their housings are aligned and fixed into position off-axis with the GRIN lens. The two housings are placed

inside a third (outer) housing made of stainless steel, and the back end of the housing is covered with a heat-shrink

tubing. The two free ends of the optical fibers were terminated with FC/PC-type male connectors for easy mating

with the laser/detectormodule comprised of a temperature-controlled laser diode (Toshiba model TOLD 9215), a
 4 NASA TM–107120

Semi-conductor laser

Monomode optical

fiber for receiving

Heat shrinking

tubing

Monomode

optical fiber for

beam transmission Outer

housing

GRIN

lens

Fiber

housing Lens housing

Air gap

Fiber

ferrule

Digital correlator Avalanche photo

diode (APD) detector

Flexible

tubing Optical head

(DLS probe)

Dime

Miniaturized

microscope

from Olympus

Teflon tubing

Imaging system

Figure 1.—A compact optical probe for monitoring protein crystallization.

Protein

droplet

constant power driver, a photomultiplier tube (PMT) (Hamamatsu HC-120) and an amplifier-discriminator system

(Brookhaven Instruments Corporation, NY). An avalanche photo diode (APD) photon counting module (model

PCS-2, EG and G Canada) was also used as a photodetector. The PMT and the APD were used to collect scattered

light from concentrated polystyrene solutions and protein solutions, respectively.

Our optical design (probe) provides major improvements over previously reported DLS systems. These improvements include accurate determination of particle sizes from very small (3 nm) to very large (~1 μm) with very

short data acquisition times (within seconds) and extremely small laser power levels (few μW) from a laser diode,

and the ability to place the probe outside the sample container anywhere from 0 to 17 mm from the sample's surface.

The probe can also be immersed in the solution. Another major improvement is its ability to probe solution dynamics

in sample containers of varying size and shapes. We have performed accurate and reliable particle size measurements with this probe on dispersions contained in regular spectrographic cuvettes, capillary tubes, common laboratory utensils (beakers, graduated cylinders, conical flasks, glass/clear plastic container vessels), concentric cells

containing two different solutions and hanging fluid drops. Extremely small sample quantities of protein solutions

(~30 μL) were interrogated in the experiments reported in this paper. Ansari and Suh (ref. 12) have recently shown

that the performance of their probe is far better when compared with the lensless probe of Dhadwal et al.

(ref. 9). The newly designed fiber optic probe was thoroughly tested using polystyrene monodisperse standards ranging in size from 20 nm to 0.8 μm at several concentrations and bovine serum albumin (BSA) protein samples ranging

in concentration from 2 to 10 percent (w/ν). These measurements were performed in batch mode and in hanging

drops and were recently reported by Ansari and Suh (ref. 12). The effect of data acquisition time (experiment duration) and the incident laser power using the probe was also tested with regard to yielding correct particle sizes. The

results showed that accurate particle size measurements were obtainable in 5 s at a laser power of 0.2 mW. Such

short measurement times may be useful for studying nucleation and growth phenomena. Recently, Ansari et al.

(ref. 6) have used this probe to detect early signs of eye diseases (e.g., cataracts) in live animals and cadavers.

Experimental Set Up and Procedure

The experimental set up was comprised of four parts; a temperature-controlled laser/detector module, a protein

crystal growth facility (cuvette, HDVDA and DCVDS), a fiber optic probe and a data acquisition system. The experimental configurations are shown in figures 2 and 3. The probe was mounted on the HDVDA/DCVDS using a

V-clamp and an optical post. Laser light (λ = 670 nm) from a laser diode at 80 μW power level was launched into the

sample via the probe. Light scattered from the particles inside the scattering volume (~2×10–5 mm3) was collected at

a scattering angle of 161.5° by the same probe assembly and was guided to the PMT or APD. The resulting signal
NASA TM–107120 5

Six-pack HDVDA

DLS probe

Plunger

Modified HDVDA for

temperature-induced

crystallization Hanging

drop

Thermocouple

Optical window

30 μl

drop

Double barreled

syringe assembly

Figure 2.—Experimental setup: hanging drop vapor diffusion apparatus (HDVDA) configuration.

Optical post

Laser

Detector

To

video

Cell body Cap

Quick disconnect

couplings on each

line allows for

individual cell and

sample retrieval

New fiber

optic DLS

probe

22 mm glass

coverslip with a

drop hanging

Gas flow lines

Probe holder

Slip

plate

30 μl

hanging

protein

droplet

Thaumatin protein crystals

Top view:

Protein drop

Cover plate

DCVDS protein

crystal growth

chamber

New fiber

optic DLS

probe

Figure 3.—Experimental setup: dynamic control vapor diffusion system (DCVDS) configuration.
 6 NASA TM–107120

was fed to a digital correlator card (Brookhaven Instruments BI9000) housed in a docking station for a PentiumTM

based notebook computer (7600 Series, EPS). A logarithmic correlation time scaling was used to capture the dynamics of very small and very large particles which may be present in the same scattering volume. Another unique feature of our DLS probe is that it is equipped with a miniaturized microscope which can be used for visualizing

macroscopic particles once they appear during a crystallization experiment. However, in the experiments reported in

this paper this feature was not utilized in on-line configuration because of inadequate white-light sample illumination conditions in the experimental chambers.

The first apparatus tested was a modified HDVDA. This apparatus, shown in figure 2, is a modification of space

flight hardware which mimics the hanging drop vapor diffusion method for growing protein crystals. The HDVDA

is equipped with an optical window made of plexiglass. This window has a thickness of 3.8 mm and the distance

from the front of the hanging droplet to the back surface of the window is 2.5 mm. In the HDVDA, the growth solution is suspended from a double barreled syringe which is surrounded by a wicking material containing the reservoir

solution. Initial solution concentrations are chosen such that water diffuses from the droplet into the reservoir. In the

modified HDVDA, the wicking material was removed and the droplet chamber size was reduced (fig. 2). Thermoelectric devices were embedded around the chamber so that temperature induced crystallization could be performed

instead of vapor diffusion. The protein solution was placed in one side of the double barreled syringe and the crystallizing agent in the other side. The syringe was then placed in the HDVDA and the two solutions were deployed

together as a droplet.

The second apparatus used was a dynamically controlled vapor diffusion system (DCVDS), shown in figure 3,

which also mimics the hanging drop vapor diffusion method. This device, however, uses controlled sequential

purges of a dry nitrogen gas to evaporate water from the growth solution at a user prescribed rate. This approach

replaces the reservoir solution used in the traditional HDVDA for accomplishing vapor diffusion. A humidity sensor

is used to monitor the amount of water evaporated out of the growth solution as a function of time. The feedback

from this sensor can be used to effect virtually any evaporation profile. A microcomputer utilizing custom software

and data acquisition/interface boards operates and monitors all aspects of the system.

Sample Preparation

A 40mM sodium acetate buffer solution was prepared at pH 4.3 and was used to dissolve lysozyme from

Boehringer to a concentration of 38.2 mg/ml. The same buffer was used to prepare a 7 percent (w/ν) NaCl stock

solution used as a crystallizing agent. Thaumatin from sigma was dissolved in 50mM ADA buffer at pH 6.5 to a

concentration of 68.5 mg/ml. The same buffer was used to prepare a stock solution of NaK tartrate at 0.075 M. All

solutions were filtered through 0.2 μm Acrodisc filters.

RESULTS AND DISCUSSION

Detection of the Onset of Temperature-Induced Aggregation in a Modified HDVDA.—We first tested the feasibility of our probe to detect the onset of temperature-induced aggregation in a 25 mg/ml solution of lysozyme dissolved in 40mM acetate buffer, pH = 4.3, with 2.5 percent w/ν NaCl. These experiments were conducted in a

modified HDVDA as shown in figure 2. The average particle size and the average scattered intensity (countrate)

were monitored simultaneously as a function of time as shown in figure 4(a). The method of cumulants was used

to reduce the DLS data to particle size. We distinguish three distinct regions in this plot. The experiment temperature began at 27 °C and was kept constant for 1 hr. The data was collected every 60 sec for a duration of 30 sec. At

stage 1 (up to 1 hr) the average scatted intensity and average size of the protein molecules remained essentially constant (intensity within 1.34 percent and size within 2 percent). After this time the solution temperature was ramped

down to 16 °C at a rate of 1 °C/min. At stage 2 both the average scattered intensity and average particle size increased, and we interpret this as an indication of the onset of protein aggregation. This behavior corroborates the

earlier observations of Casay and Wilson (ref. 11) on the basis of their DLS and static light scattering measurements.

The temperature was then ramped up to 21 °C at 1 °C/min and maintained until the total experiment time was about

3 hr. At stage 3 we note a reduction in average size of the particles and average scattered intensity indicating some

degree of dissociation of the primary aggregates. Data selected from the three stages was analyzed using CONTIN

and is plotted in figure 4(b). The histogram presentation shows the shifts in average particle size corresponding to
NASA TM–107120 7

stages 1 to 3 and is consistent with the cumulant analysis data shown in figure 4(a). A polydispersity factor, described in reference 4 and measured from the DLS-TCF data, can be a sensitive measure of aggregation. In our experiments, this factor, often referred to as the poly, increased from 0.005 in the initial stage to 0.22 in the later state

suggesting a change from a nearly monodisperse state to a more polydisperse state. The state of protein aggregation

corresponding to stage 1 is clearly best described as that of lysozyme monomer since the average particle diameter

was determined to be about 3.8 nm and the poly value was extremely low. At stage 2, the average diameter increased to between 5 and 6 nm with an accompanying increase in poly. The actual particle size distribution at stage

2 is indeterminate from these data, and no inferences concerning specific monometer ↔ n-mer equilibria are made.

However, from these results we do conclude that our newly designed probe is quite sensitive for detection of the

aggregation stages in a dynamically controlled protein solution environment.

Detection of Fast Crystallization in the HDVDA and DCVDS.—The results described in the previous section

(section 5.1) were for lysozyme solutions at relatively low supersaturation. The data analysis showed that the aggregates detected were small with no indication of particle sizes greater than 10 nm and certainly nothing of microscopic Diameter (nm)

8

6

4

2

0

0.0 3.0 0.6 1.2 1.8 2.4

Time (hr)

Stage 1

Stage 2

Stage 3

Countrate (kc/s)

180

170

160

150

140

Diameter

Countrate

(a)

4-a

100

Intensity

80

60

40

20

0

1 10 2 3 4 5 6 7 89

Stage 1

Stage 2

Stage 3

Diameter (nm)

(b)

4-b

Figure 4.—Detection of the onset of temperature-induced aggregation in

 a hanging drop of lysozyme solution. (a) Average particle size and

 average scattered intensity as a function of time. (b) Size distribution.
 8 NASA TM–107120

size. But what about the case when high protein supersaturations are employed? Crystallization in some proteins can

be induced relatively quickly with large populations of microcrystals formed. In order to test the latter case, two

experiments were performed utilizing supersaturated lysozyme in the HDVDA and supersaturated thaumatin in the

DCVDS. The first experiment tested the probe on a supersaturated solution of 85 mg/ml lysozyme in 40mM acetate

buffer, pH = 4.3 with 4 percent NaCl. The lysozyme solution was loaded in the six-pack HDVDA unit at an initial

temperature of 24 °C. The average particle size and average scattered intensity as a function of time is plotted in

figure 5. The initial particle sizes in this solution are dramatically different from that depicted in figure 4(a). This

solution clearly contained particles that had an average diameter of several hundred nm, not surprising for such a

highly supersaturated solution. The temperature, 24 °C, was held constant for about 40 min during which the average particle size was maintained. After about 40 min the temperature of the lysozyme solution was quickly reduced

to 21 °C. Immediately upon cooling, both average size and average scattered intensity increased and finally,

lysozyme crystals (~0.1 to 0.2 mm) appeared. This general trend in the increase in particle size has been noted by

Malkin and McPherson (ref. 13) in a light scattering study of STMV, apoferritin and pumpkin globulin at several

different high supersaturation levels. Figure 6(a) shows typical TCF profiles at the initial, intermediate and crystallizing stages of the solution. The first TCF (t = 0), an intermediate TCF (t = 40 min) and last TCF (t = 55 min) show

significant change in their shape. This is reflected in the histogram presentations of particle size shown in figure

6(b). The initial particles (t = 0) had sizes centered around 5 nm and 0.5 μm while the particles at the crystallizing

stage (t = 55 min) showed two sizes; one centered around 8 nm and another 5 μm. These measurements indicate the

sensitivity of the DLS probe for detecting the presence of both small and large particles in the same solution. We do

not wish to emphasize the absolute values for the particle size distributions, but instead, point out the good dynamic

range of the DLS probe.

1000

100

0 60 12 24 36 48

Time (min)

10 000

Diameter

Intensity

3000

2000

1000

0

Intensity (kc/s)

24 °C

Average diameter (nm)

21 °C

Figure 5.—Temperature-induced crystallization in a lysozyme system in

 a six-pack HDVDA.

DLS measurements were also performed on another supersaturated solution comprised of 34.35 mg/ml thau–

matin mixed with 0.038M NaK tartrate in the DCVDS at the highest rate of evaporation. The TCF profiles are

plotted in figure 7. The t = 0 measurement shows a particle size centered around 5 nm which can be regarded as

thaumatin monomers. After 4 min of evaporation the size distribution increases very rapidly centering around

4.3 nm and 13 μm. Ten minutes later the solution was examined again. It showed a size distribution centered

around 4.7 nm suggesting sedimentation of large crystals. Indeed upon examination under a microscope the crystals (~0.1 to 0.2 mm) appeared at the bottom of the hanging thaumatin droplet and are shown in figure 3. Again,

we have demonstrated the effectiveness of the DLS probe for detecting large changes in particle size that can be

forced to occur in a protein crystallization experiment.
NASA TM–107120 9

1.00

0.60

0.40

0.00

5 2000 10 100 1000

Delay time (micro sec)

0.80

Normalized intensity autocorrelation 0.20

(a)

Initial A

Intermediate B

Crystallization C

C

B

A

6-a

100

60

40

0

1 10 000 10 100 1000

80

20

(b)

Intensity

Diameter (nm)

Initial

Intermediate

Crystallization

Figure 6.—Profiles representing various stages of crystal growth in the

 lysozyme system used in Figure 5. (a) Time correlation function (TCF).

 (b) Size distribution.

6-b
 10 NASA TM–107120

CONCLUSION

We conclude that noninvasive, quantitative, fast, and reliable DLS measurements are now possible in hanging

drop configurations suitable for ground-based and microgravity protein crystal growth facilities. The newly designed probe is capable of detecting the various particle sizes which may be associated with all of the various stages

of protein crystal growth.

ACKNOWLEDGMENTS

The support of the Microgravity Science and Applications Division (Code UG) of the NASA headquarters is

thankfully acknowledged. The probe was developed by R.R. Ansari and K.I. Suh at the NASA Lewis Research

Center under grant number NCC 166-3/CWRU (Task Number 1566). The HDVDA system was developed by

L.J. DeLucas and others and the DCVDS was developed by T.L. Bray and L.J. DeLucas at the UAB/CMC. The

modified HDVDA for temperature-induced studies was developed by W.W. Wilson of MSU. KI. Suh would like

to thank National Research Council (NRC) and the NASA Lewis Research Center of the award of a postdoctoral

research fellowship.

1.00

0.60

0.40

0.00

5 2000 10 100 1000

Delay time (micro sec)

0.80

Normalized intensity autocorrelation 0.20

(a)

Pre aggregation (monomeric state)

Upon crystallization

Post crystallization (~ monomer)

7-a

100

60

40

0

1 10 000 10 100 1000

80

20

(b)

Intensity

Diameter (nm)

Pre aggregation (monomeric state)

Upon crystallization

Post crystallization (~ monomer)

Figure 7.—Profiles representing various stages of crystal growth in a fast

 crystallizing thaumatin system. (a) TCF. (b) Size distribution.

7-b
NASA TM–107120 11

REFERENCES

1. Littke, W. and John, C., Science 225, p. 203, 1984.

2. Rosenblum, W.M., DeLucas, L.J., and Wilson, W.W., Protein Crystal Growth in Microgravity, in: Proc.

NASA Laser Light Scattering Advanced Technology Development Workshop, NASA CP–10033,

September, 1988, pp. 235–256.

3. Bray, T.L., Kim, L.J., Askew, R.P., Harrington, M.D., Rosenblum, W.M., Wilson, W.W., and

DeLucas, L.J., New Crystallization Systems Envisioned for Microgravity Studies, J. Appl. Cryst.,

submitted.

4. Chu, B., Laser Light Scattering: Basic Principles and Practice (Academic Press, New York, 1991).

5. Ford, N.C., Theory and Practice of Correlation Spectroscopy, in: Measurements of Suspended Particles by

Quasi-Elastic Light Scattering, Ed., Dahneke, B.E. (Wiley-Interscience, New York, 1983) pp. 31–78.

6. Ansari, R.R., Suh, K.I., DellaVecchia, M.A., and Dubin, S., Ophthalmic Diagnostics Using a New Dynamic

Light Scattering Fiber Optic Probe, in: Proc. Medical Application, Conf. on Lasers in Ophthalmology III,

September, 1995, Barcelona, Spain, to be published in Proc. SPIE–2632, No. 18.

7. Stock, R.S., and Ray, W.H., J. Polym. Sci.: Polym. Phys. Edition 23, p 1393, 1985.

8. Brown, R.G.W., Appl. Optics 26, 1987, p 4846; also Appl. Opt. 29, p. 4159, 1990.

9. Dhadwal, H.S., Method and Apparatus for Determining the Physical Properties of Materials Using Dynamic

Light Scattering Techniques, U.S. patent No. 5,155,549, October 13, 1992.

10. Dhadwal, H.S., Ansari, R.R., and Meyer, W.V., Rev. Sci. Instrum 62 (12), p. 2963, 1991.

11. Casay, G.A., and Wilson, W.W., J. Crystal Growth 122, 1992, p. 95.

 12 R.R. Ansari and K.I. Suh, Sizing of Colloidal Particles and Protein Molecules in a Hanging Fluid Drop, in:

Proc. Biomedical Optoelectronics in Clinical Chemistry and Biotechnology, 12–16 September 1995,

Barcelona, Spain, to be published in Proc. SPIE 2629, No. 23.

13. Malkin, A.J., and McPherson, A., Acta Cryst. D 50, 1994, p. 385.
REPORT DOCUMENTATION PAGE

2. REPORT DATE

19. SECURITY CLASSIFICATION

 OF ABSTRACT

18. SECURITY CLASSIFICATION

 OF THIS PAGE

Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources,

gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this

collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson

Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget, Paperwork Reduction Project (0704-0188), Washington, DC 20503.

NSN 7540-01-280-5500 Standard Form 298 (Rev. 2-89)

Prescribed by ANSI Std. Z39-18

298-102

Form Approved

OMB No. 0704-0188

10. SPONSORING/MONITORING

 AGENCY REPORT NUMBER

8. PERFORMING ORGANIZATION

 REPORT NUMBER

5. FUNDING NUMBERS

1. AGENCY USE ONLY (Leave blank) 3. REPORT TYPE AND DATES COVERED

4. TITLE AND SUBTITLE

6. AUTHOR(S)

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)

9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES)

11. SUPPLEMENTARY NOTES

14. SUBJECT TERMS

17. SECURITY CLASSIFICATION

 OF REPORT

16. PRICE CODE

15. NUMBER OF PAGES

20. LIMITATION OF ABSTRACT

Technical Memorandum

Unclassified Unclassified Unclassified

National Aeronautics and Space Administration

Washington, DC 20546–0001

National Aeronautics and Space Administration

Lewis Research Center

Cleveland, Ohio 44135 – 3191

12a. DISTRIBUTION/AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE

13. ABSTRACT (Maximum 200 words)

NASA TM–107120

E–10016

November 1996

14

A03

A Fiber Optic Probe for Monitoring Protein Aggregation, Nucleation,

and Crystallization

Rafat R. Ansari, Kwang I. Suh, Alireza Arabshahi, W. William Wilson,

Terry L. Bray, and Lawrence J. DeLucas

WU–963–45–0A

NCC 166–3

This publication is available from the NASA Center for Aerospace Information, (301) 621–0390.

Protein crystals are experimentally grown in hanging drops in microgravity experiments on-board the Space Shuttle orbiter. The

technique of dynamic light scattering (DLS) can be used to monitor crystal growth process in hanging droplets (~30 (L)) in microgravity

experiments, but elaborate instrumentation and optical alignment problems have made in-situ applications difficult. In this paper we

demonstrate that such experiments are now feasible. We apply a newly developed fiber optic probe to various earth and space (microgravity) bound protein crystallization system configurations to test its capability. These include conventional batch (cuvette or capillary)

systems, hanging drop method in a six-pack hanging drop vapor diffusion apparatus (HDVDA), a modified HDVDA for temperatureinduced nucleation and aggregation studies, and a newly envisioned dynamically controlled vapor diffusion system (DCVDS) configuration. Our compact system exploits the principles of DLS and offers a fast (within a few seconds) means of quantitatively and noninvasively monitoring the various growth stages of protein crystallization. In addition to DLS capability, the probe can also be used for

performing single-angle static light scattering measurements. It utilizes extremely low levels of laser power (~few (W)) without a need

of having any optical alignment and vibration isolation. The compact probe is also equipped with a miniaturized microscope for

visualization of macroscopic protein crystals. This new optical diagnostic system opens up enormous opportunity for exploring new

ways to grow good quality crystals suitable for x-ray crystallographic analysis and may help develop a concrete scientific basis for

understanding the process of crystallization.

Prepared for the Sixth International Conference on Crystallization of Biological Macromolecules sponsored by the Hiroshima Prefecture,

Hiroshima City, the Ministry of Education, Science, Sports and Culture, and the International Union of Chrystallography, Hiroshima, Japan,

November 12–17, 1995. Rafat R. Ansari, NASA Lewis Research Center; Kwang I. Suh, National Research Council—NASA Research

Associate at Lewis Research Center; Alireza Arabshahi and W. William Wilson, Mississippi State University, Department of Chemistry, P.O.

Drawer CH, Mississippi State University, Mississippi 39762; Terry L. Bray and Lawrence J. DeLucas, University of Alabama at Birmingham,

Center for Macromolecular Crystallography, Birmingham, Alabama 35294. Responsible person, Rafat R. Ansari, organization code 6712,

(216) 433–5008; fax 216-977-7138; email ransari@lerc.nasa.gov.

Subject Categories 35, 51 and 74

Unclassified-Unlimited

Dynamic light scattering; Protein crystal growth; Droplet; Particle sizing; Microgravity