Patent Application
20080225265
1. Field of the Invention
The present invention pertains generally to the field of macromolecular characterization, and more particularly to the field of determining crystallization conditions related to a macromolecule.
2. Background of the Invention
Macromolecules include proteins, protein complexes, enzymes, nucleic acids, viruses, and generally any large complex molecule. Macromolecules find a wide range of applications, from pharmaceuticals to enzymes for medical diagnostic or industrial use. Macromolecules are almost always the targets for the development of new pharmaceuticals.
A critical step in the understanding of the function and operation of a particular macromolecule is to determine the macromolecular structure. Tools for determining structure, such as x-ray diffraction crystallography require a crystallized sample of the macromolecular material. The process for producing a crystallized sample typically involves obtaining a DNA sequence encoding the macromolecule, cloning and expression to generate a sufficient quantity of sample, purification to remove interfering substances, and finally crystallization. Since each of these steps is complex, only a limited number of targeted macromolecules, in particular, proteins reach the structure determination stage, and for those that do, only a very small sample of material may be available for analysis.
Crystallization of macromolecules is a delicate process requiring just the right concentration, compatible solution, and temperature. Macromolecule crystallization trials are typically carried out using a widely varying array of crystallization solutions, or ‘cocktails’, typically in blocks of 96 solutions at a time. The solutions are generated from a potential search space of dozens to hundreds of potential ingredients with a wide range of concentrations for each ingredient, together with pH and temperature variables. The number of permutations of solution definition characteristics is daunting. The results from these trials are then typically interpreted in a yes/no manner, i.e., crystal or no crystal. The data provides little guidance for subsequent trials unless a crystal is actually formed. Thus, many trials and/or macromolecule modifications at the chemical or genetic level may be required before the proper crystallization conditions are determined.
Thus, there is a need for a system and method for determining crystallization conditions of a macromolecular material that reduces the search space and potentially finds crystallization conditions rapidly, in a minimum number of trials, and needs only a small sample of the material.
Briefly, the present invention pertains to a system and method for determining macromolecule crystallization conditions by measuring the polarization anisotropy of a fluorescent probe attached to the macromolecule in solution as a function of a variation in crystallization conditions. In one exemplary embodiment, the concentration of the macromolecule material is varied and the polarization anisotropy as a function of concentration gives an indication of the proximity to crystallization conditions. A pulse illumination system with time gated detection is disclosed to isolate fluorescence response from excitation to reduce noise due to scattered and reflected light. A microassay system is disclosed to allow a complete 96 condition screen with less than 1 micro-liter of solution.
These and further benefits and features of the present invention are herein described in detail with reference to exemplary embodiments in accordance with the invention.
The present invention is described with reference to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the left-most digit(s) of a reference number identifies the drawing in which the reference number first appears.
Finding crystallization conditions for macromolecules is presently a tedious trial and error process where numerous cocktails of solvents, solutes, and the macromolecules are tested over a range of parameters such as concentration, temperature, and pH to find a set of conditions for crystallizing the macromolecule material. Success or failure is pinned on finding a crystal in a sample. The present invention streamlines the process by observing subtle changes in macromolecule solution properties that indicate a greater propensity to form a crystal. By observing these properties, a sample that does not yield a crystal and thus would yield a negative result in the conventional method may yield a measurement indicating a potential propensity for crystallization and thus point the way for further tests using related conditions to efficiently converge on the right conditions for crystallization.
Crystallization is a self association process where the molecules sequentially arrange themselves in an orderly manner. For macromolecules, there is a narrow range of attractive interaction strengths, known as the crystallization slot (references 1, and 2), that favor the crystallization process. If the interaction forces are too strong, non crystalline precipitate is obtained. If the interaction forces are not strong enough, or are repulsive, then a clear solution is obtained.
In accordance with the present invention, the strength of interaction forces is determined by measuring the fluorescence anisotropy using a fluorescent tag (alternatively referred to as a probe) attached to the macromolecule. By observing changes in the interaction forces over a set of conditions, the more favorable crystallization conditions may be identified. In accordance with the present invention, a sample is illuminated with polarized light at the excitation wavelength of the tag and the polarization of the fluorescence is observed for an indication of the rotation rate of the molecule in solution. The illumination is absorbed most favorably in certain orientations of the molecule and the emission is related in polarization to the polarization of the absorbed illumination, but may be rotated as the molecule rotates in solution due to random thermal motion. The rotation rate in turn, will be influenced by molecule size and will be reduced as attraction forces between molecules increase and possible temporary molecule pairs may form. Thus, by observing the anisotropy of the polarization of the fluorescence emission, the average rotation rate may be observed, indicating the tendency to form crystals. To find conditions favorable for crystallization, the polarization anisotropy may be observed for a set of variable conditions and the most favorable conditions determined from evaluation of the observed rotation rates. Increasingly favorable conditions may be found by varying new conditions based on previously found most favorable conditions. Thus, useful information leading to finding crystallization conditions may be found from conditions that do not yet yield crystals—leading to the finding of crystallization conditions with many fewer trials.
By using pulsed illumination and time gating of the fluorescence signal scattered light from the illumination can be eliminated. This scattered light can be responsible for a considerable amount of random variability or noise in the fluorescence signal. Since the fluorescence is orders of magnitude less than the illumination, the scattered light from the illumination is difficult to eliminate by filters alone. Time gating allows for elimination of the illumination response. The time gating thus allows for greater toleration of contamination which, along with other higher molecular weight species present such as some precipitants, is responsible for scattered light and allows for smaller test volumes due to the improved signal to noise which allows use of a reduced fluorescence signal from the smaller volume.
In a further benefit, the technique is relatively insensitive to absorption from contaminants and other sources. The anisotropy measurement is a ratiometric measurement, depending only on the ratio of two components of fluorescent emission and is independent of the incident intensity. Thus, variation in factors such as the source intensity, or absorption by components or contaminants in the solution will have minimal effect on the anisotropy measurement.
Traditional methods for measuring the strength of interaction for crystallization conditions typically use light scattering (reference 1) and self-interaction chromatography (references 2 and 3). These methods are not well suited for making a large number of measurements on a small volume of solution. The light scattering method, in particular, is highly susceptible to noise and interference from other large molecules in the solution. Self interaction chromatography suffers from having to prepare a column matrix with covalently attached protein, having to then pour and calibrate the analytical column, and having to reequilibrate the column with each test precipitant solution of interest.
Prior to the measurements, proteins need to be prepared properly. The protein is covalently labeled with a suitable fluorescent probe, using methods well known to those familiar with protein modification chemistry. The preferable sites for labeling, in order, are the N-terminal amine, randomly labeled amine side chains, free sulfhydryl groups, and random carboxyl groups. Typical labeling procedures are provided in the literature or otherwise known in the art. Other sites may be labeled using the appropriate labeling chemistry and probes.
The labeling procedure typically involves:
The fluorescent probe concentrations in the assay should be between 10e-8 and 10e-6 M, with the fraction of protein molecules labeled typically being around 1%. This fraction is calculated based upon the protein's molecular weight and assumes a stock protein concentration for crystallization screening of 10 mg/ml.
The anisotropy r is calculated with these intensity measurements as:
where,
IVV is the component parallel to the incident (excitation) illumination, and
IVH is the component perpendicular to the incident illumination.
For one exemplary tag, the anisotropy value varies from 0.4 when the probe's absorption and emission polarization vectors are parallel, to −0.2 when the absorption and emission vectors are at a right angle. Since the anisotropy is a property of the structure of the fluorescing species other species may have different values.
Fundamental anisotropy, r0, may be determined with the fluorescing species held stationary in a glass or frozen medium such that the molecules cannot rotate. When the molecules can freely rotate as in a liquid solution, the anisotropy is a function of time since excitation because of the rotation of the molecules due to random thermal motion. The anisotropy will be initially r0 as measured in the glass medium, but will decay with time to zero (isotropic) as the molecules randomize. Measured anisotropy is thus a function of the measurement time related to the rotation rate of the molecules.
The anisotropy is a function of rotational correlation time, Θ;
where r0 is the fundamental anisotropy of the fluorescing species, and τ is the fluorescence life time (time to 1/e intensity, where e is the natural logarithm base). The rotational correlation time is, in turn, a function of molecular weight, M, of the macromolecule or macromolecule assembly as.
where η is the viscosity of the solution, R is the ideal gas constant, T is the absolute temperature, v is the specific volume of the macromolecule, and h is the hydration. For a given screening solution all parameters in the equation 3 but M typically stay constant. Note that hydration may reduce as water is lost at molecular contact sites, but the amount should be a relatively small change. Also, when comparing solutions of different viscosities, note that in equation 3, a change in η has the same effect on Θ as a proportionally equivalent change in M, thus it is important to account for solution viscosity when estimating changes in M by anisotropy measurements. In some cases, temperature, T, may be varied to determine optimum temperatures for crystallization.
Thus, in the typical solution, the increase of M is an indication of the macromolecule self associating, with the rate of increase of anisotropy as a function of the concentration in a given solution being characteristic of the form of self association, i.e., structured or non-structured, crystal or non-crystal.
This method enables us to measure increase of M by the increase of r. In other words, measuring anisotropy to monitor macromolecule self-association. In one embodiment, the fluorescent tag is selected to have a lifetime (decay time) commensurate with the rotation correlation time. Preferably, the rotation correlation time of a single molecule is shorter than the lifetime so that, as the mass increases by the association of two or more molecules, the anisotropy increases toward mid range and above.
The shape of the plots may be observed to estimate the likelihood of crystallization. An ideal curve begins with low anisotropy and increases gradually and monotonically, although slight up and down variation due to measurement noise and experimental variation is tolerated. Experimental variation may arise from several sources including variation in solution preparation, variation in contamination or surface effects of the containers. Measurement noise may include any noise source in the illumination or detection process including timing variation and statistical noise in counting photons. Measurement noise and experimental variation may be determined from each experimental apparatus by observing variations in a large number of samples, especially non-trending samples. The inventors have observed 5% variation in one experimental apparatus, i.e. 5% of the anisotropy span from zero to r0.
At some concentration level, the curve begins a rapid acceleration upward indicating a tendency to associate. In contrast, a high value for low concentrations suggests strong attraction forces that lead to noncrystalline or microcrystalline precipitate. Also a decrease with increasing concentration suggests a solution that will not crystallize.
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The light source should be a monochromatic or narrow bandwidth short pulse. Both lasers and light emitting diodes are suitable for this requirement. Although lasers have narrower waveband and the beam is easier to be condensed, lasers are more expensive and more difficult to operate. Newer LED's may offer comparable performance with greater ease of use.
An exemplary LED is the Nichia NSPB300A, or LumiLED Superflux, or other high intensity LED having a relatively narrow emission angle and spectrum at the desired excitation wavelength.
However, the rotational rate is also proportional to the solution viscosity. High viscosity precipitant solutions, such as those having 25% or 30% Polyethylene Glycol (PEG), will give higher anisotropy values even at low protein concentrations. In principle, the fluorescent probe should have a lifetime commensurate with the anticipated rotational rate of the molecule to be measured.
Referring to
When comparing solutions that have differing viscosities, the viscosity dependence can potentially cause confusion. For most proteins this would result in an increase in the measured anisotropy, and the data would appear to indicate that the protein is precipitating, that the conditions are not conducive to crystallization.
Use of very long lifetime probes also means that for smaller proteins there is very little change in anisotropy during the early stages of crystal nucleation. This problem can also be reconciled by recognizing that we are attempting, first and foremost, to eliminate conditions that lead to rapid precipitation from further consideration. By using fluorescent probes with very long lifetimes we can still collect data along the bottom of the anisotropy curve, i.e. begin with low concentrations at low anisotropy—nearly isotropic. Thus, mono dispersed protein molecules have plenty of time to randomize their positions before the data acquisition is completed. Dimerized and larger associations of molecules will be less random and show slightly elevated anisotropy values. Precipitated protein will have a large apparent mass, and thus have high anisotropy values (close to r0) even at low concentrations. High viscosity solutions of mono dispersed protein will result in elevated anisotropy values, but these will be well below the limiting value (r0) and, in the case of crystallization, still show an expected progressive rise with concentration if pre-crystalline self association is taking place. Low molecular weight mono dispersed solutions having low viscosity will also show a slight rise in anisotropy value with increasing concentration of the protein.
The fluorescence probes of choice are Metal Ligand Charge Transfer (MLCT) complexes, such as ruthenium bis(2,2′-bipyridine)-4,4′-dicarboxybipyridine (Ru(bpy)2(dcbpy)). This probe has an excitation wavelength (Exmax) peak at around 460 nm and an emission wavelength (Emmax) peak at around 630 nm, the fluorescence lifetime σ is around 400 nano-seconds, and the fundamental anisotropy r0 is 0.26 at 485 nm. Having a long lifetime, the fluorescence energy conversion is relatively inefficient. The fraction of light absorbed per mole, ε=14,500 M−1, and the quantum yield is about 0.05. However, the large stokes shift (difference in wavelength between Exmax and Emmax) facilitates removal of the excitation from the emitted light by applying a low-pass filter, while the long lifetime enables removal of short lived noise (scattering and reflections of the excitation light, and any intrinsic fluorescence from the sample) by applying time gating. Other long lifetime fluorescent probes will also be suitable.
This probe (Ru(bpy)2(dcbpy)) is commercially available as an amine-reactive activated disuccinimidyl ester. A number of other Ru-based probes may also be used. MLCT's based on Rhenium (Re) and Osmium (Os) have also been described [ref. 5]. The Re based probes typically have longer lifetimes, higher quantum yields, and blue-shifted excitation and emission spectra relative to Ruthenium (Ru) based probes, while the Os based complexes typically have shorter lifetimes and red shifted spectra. The Re complexes in particular are often oxygen sensitive, but this sensitivity typically decreases due to shielding upon conjugation to a protein. [Ru(bpy)2(dcbpy)] typically shows good absorption of excitation energy below 500 nm with a peak around 450 nm. The absorption slightly improves when conjugated to Human Serum Albumin (HSA), and the peak shifts to around 460 nm.
The anisotropy may also be characterized as a function of the excitation wavelength and bonding state. When conjugated with HSA, the probe has good anisotropy from about 460 nm to 510 nm. Thus, a good excitation wavelength may be around 480 nm where the probe has a weak but usable absorption efficiency and gives good anisotropy.
The emission spectrum of [Ru(bpy)2(dcbpy)] conjugated with HSA shows a peak around 650 nm with virtually no emission shorter than 550 nm. Note that the emission spectrum is well separated from the 480 nm excitation. Thus, a wavelength filter or dichroic mirror may be used to separate the excitation energy from fluorescence response energy.
Curves 607 through 609 are where additional crystallization conditions may be found that would not be recognized as such using current methods. In a standard screening methods where crystals are the desired endpoint, the outcomes at these conditions would likely be interpreted as either micro-granular or amorphous precipitate and considered to be failure. However, the low concentration anisotropy data indicates that the protein is showing a concentration-dependent self association. Therefore, we propose that if one can reduce the strength of the interactions the curves could be shifted to the right, such that they were more like curves 604 and 605. This can be brought about by reduction in the concentration(s) or composition of the precipitant solution components, and/or by the use of additives.
Additives are commonly employed in protein crystallization. Many additives act by increasing the solubility, which would have the effect of shifting the curves to the right. Testing for suitable additives can also be carried out using the anisotropy approach, and may not need a full titration curve, but only one data point. For example, if the “stock” condition gives anisotropy values at r0 at, say 0.12× dilution, then addition of an additive and finding an anisotropy of, for example, 0.035, would suggest that the curve has been shifted to within the potential crystallization regime.
Curve 610 is postulated as the result that would be obtained in the case of a phase separation, where the protein is crowded due to partitioning, but does not undergo any further self association.
The sample tray 109 is located on an X-Y stage for precise movement in X 714 and Y 712 directions. The movement of the tray 708 and sample distribution heads 702 has to be accurate enough to locate the drops onto the same spots 710 for proper mixing. Accuracy of a few microns may be necessary for the smallest samples. Mixing is by diffusion over the short distances of the 1 to 10 nanoliter volume drops. The sample array may then be moved to the measurement optics 706 and the tray 708 moved to each sample 710 in turn as data is collected. In an alternative embodiment, the optics 706 may move and the tray 708 may remain stationary.
Merit function
The merit function may be based on one or more of the following:
1. the anisotropy value at the lowest sample concentration,
2. monotonically increasing anisotropy with concentration, and
3. goodness of least squares fit of defined ideal curves to the data.
The merit function calculates a weighted sum of multiple factors listed above. A preferred criterion may be based on curve fitting to a range of ideal curves. The ideal curves may be empirically determined from a number of control samples and compared with the data giving a mean square error, where zero mean square error would be a score of 100 and increasing mean square error would subtract from 100. In one embodiment, a parameter of the ideal curve may be varied to minimize the mean square error.
When comparing and ranking test results between and among different solutions, it will be desirable to correct for different viscosities as described with reference to
For each solution tested, a merit function value will be determined. If the test finds a solution with a merit function value greater than a predefined threshold value, then the test is judged successful and the system reports the resulting conditions. If the test includes no solution with an acceptable merit function value, the test procedure may be repeated with a new set of solutions. If one or more solutions show promise, but do not show a clear indication of crystallization, then the repeated test may use variations on the promising solutions. The variations may include more or less of one or more ingredients, a slight shift in pH, a slight shift in temperature, addition or subtraction of an additive or other variation. Based on the results of the variations, the specification for the solution may be further varied by extrapolation or interpolation. The results may include, but are not limited to the merit function, the trends observed, and whether precipitate or crystals were formed. Thus, through a logically developed sequence of informative iterative tests, the process may automatically follow a path to find a successful set of crystallization conditions. This iterative approach is not possible using conventional methods where the only result is whether a crystal is formed or not, with no quantitative likelihood result for the no-crystal case.
Each of the LED pulse train 908 and gating pulse train 912 may comprise any number of pulses desired to accumulate sufficient response for reliable detection, i.e., sufficient pulses to bring the detected signal above system noises to achieve the desired accuracy. The number may be for example 1000 pulses, but may be any number. Two pulses are shown. The time interval 1002 for the LED pulse may be for example 400 ns. The time 1004 after completion of the LED pulse and beginning of the gate time may be, for example 400 ns. The gate time 1006 may be for example 150 microseconds. The time 1008 to read the counter may be, for example 1 millisecond. The LED pulse is preferably on the same order or shorter than the fluorescence decay time. Longer times are less effective. The interval 1004 after the end of the LED pulse and the beginning of the PMT counting should preferably allow the LED response to fully decay and allow fluorescence from other than the tag to decay. The LED response depends on the LED selected. Typical unwanted fluorescence lifetimes will be on the order of 10 nanoseconds or less. Thus, eight to ten fluorescence lifetimes, for example, will substantially eliminate this source of noise and further improve the signal to noise ratio. The time 1006 to count the PMT pulses may be driven by energy considerations that suggest reading one or more lifetimes. Molecular rotation time considerations may suggest other time intervals. The time to read the counters 1008 is digital system dependent and may be essentially as fast as desired.
The system achieves extreme sensitivity by accumulating the response from many pulses over time, allowing a low excitation light intensity that does not disturb the solution conditions. Stray light from scattering and unwanted fluorescence is rejected by delaying the beginning of the pulse counting at a predefined time interval past the end of the excitation illumination. In a preferred embodiment this gate delay time and the data collection time are both adjustable, either through direct variation of a timing component or through a programmable setting of the timing intervals.
This disclosure is written describing in detail the use of macromolecule concentration trends to evaluate the proximity to good crystallization conditions; however, other parameters that define the solution may also be used. Macromolecule concentration is the preferred variable because it is almost universally a one way trend. Other variables may decrease or have minima or maxima that result in a more complex analysis. However several of these other parameters, such as temperature or pH or concentration of a particular component may be varied and studied for appropriate results given the variable selected. A merit factor may be used that indicates an increase in likelihood of crystallization as particle mass increases as measured using polarization anisotropy.
In particular, temperature may be used as an alternative condition parameter to be varied. Temperature is particularly convenient in that entirely new solutions need not be produced for each step. A series of solutions may be generated by using a single solution that is run through a series of temperature steps to generate a set of anisotropy measurements. The set of measurements may then be evaluated for a trend in anisotropy indicating a trend in mass as a function of temperature. A monotonically increasing trend in mass for decreasing temperature may indicate good crystallization conditions.
Thus, herein described is a system and method for determining crystallization conditions of a macromolecular material that reduces the search space and potentially finds crystallization conditions rapidly, in a minimum number of trials, and needs only a small sample of the material.
One should understand that numerous variations may be made by one skilled in the art based on the teachings herein. Such variations include but are not limited to different probes, timing, light sources, detectors, different variable conditions, such as pH, component concentration, temperature, and other factors.
The present invention has been described above with the aid of functional building blocks illustrating the performance of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed. Any such alternate boundaries are thus within the scope and spirit of the claimed invention. One skilled in the art will recognize that these functional building blocks can be implemented by discrete components, application specific integrated circuits, processors executing appropriate software and the like or any combination thereof.
While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the appended claims and their equivalents.
