Application of near-infrared spectra on temperature-controlled protein crystallization: a simulation study.

Large, high-quality protein crystals are required for the structural determination of proteins via X-ray diffraction. In this article, we propose a technique to facilitate the production of such crystals and validate its feasibility through simulations. An analytical method for protein aqueous solution based on a Fourier transform infrared (FTIR) spectroscopy is combined with a temperature control strategy to manipulate the extent of supersaturation during crystal growth, thus impacting crystal quality. The technique requires minimal knowledge about the growth kinetics a priori. The simulations show that, under ideal conditions, the design can be as effective as predesigned temperature programs for crystallization based on known growth kinetics. Two kinds of errors might be encountered with this design. Error in the estimated number of seed crystals can result in a growth rate deviating from the desired one. Nevertheless, the deviation is usually tolerable and system instability is unlikely to occur. Based on the standard error of our FTIR method, errors in concentration measurement are simulated. Measurement error can result in system instability and impair the control algorithm. Such errors may be compensated by limiting the temperature change taken by the control action, or by improving the measurement precision through the use of regressed concentrations. Through simulations, it is shown that the proposed design is practical and represents a significant improvement over the commonly used isothermal crystallization technique.

Effects of PEG on detergent micelles: implications for the crystallization of integral membrane proteins.

The solubilization of integral membrane proteins with detergents produces protein-detergent complexes (PDCs). Interactions between the detergent moieties of PDCs contribute significantly to their behavior. The effects of the precipitating agent polyethylene glycol (PEG) upon these detergent-detergent interactions have been examined, focusing on the detergent system used to crystallize the bacterial outer membrane protein OmpF porin. Static and dynamic light scattering were used to assess the effects of temperature and concentration upon the hydrodynamic size distribution and the aggregation state of detergent micelles and a phase diagram for micellar solutions was mapped. Estimates of the second osmotic virial coefficient obtained from static light-scattering measurements on micelles were shown to accurately reflect the thermodynamic quality of the solvent. Solvent quality decreases as the consolute boundary is approached, suggesting micelle-micelle attractive forces help to organize PDCs into crystalline aggregates near the cloud point. An apparent increase in micelle mass is observed as the solution approaches the cloud point. These results raise the possibility that the detergent-mediated aggregation of PDCs and/or slight changes in micelle geometry may prove to be important in the nucleation of membrane protein crystals.

The NMR structure of human beta-defensin-2 reveals a novel alpha-helical segment.

Human beta-defensin-2 (HBD-2) is a member of the defensin family of antimicrobial peptides. HBD-2 was first isolated from inflamed skin where it is posited to participate in the killing of invasive bacteria and in the recruitment of cells of the adaptive immune response. Static light scattering and two-dimensional proton nuclear magnetic resonance spectroscopy have been used to assess the physical state and structure of HBD-2 in solution. At concentrations of < or = 2.4 mM, HBD-2 is monomeric. The structure is amphiphilic with a nonuniform surface distribution of positive charge and contains several key structural elements, including a triple-stranded, antiparallel beta-sheet with strands 2 and 3 in a beta-hairpin conformation. A beta-bulge in the second strand occurs at Gly28, a position conserved in the entire defensin family. In solution, HBD-2 exhibits an alpha-helical segment near the N-terminus that has not been previously ascribed to solution structures of alpha-defensins or to the beta-defensin BNBD-12. This novel structural element may be a factor contributing to the specific microbicidal or chemokine-like properties of HBD-2.

Temperature-insensitive near-infrared method for determination of protein concentration during protein crystal growth.

A temperature-insensitive method for measuring protein concentration in aqueous solutions using near-infrared spectroscopy is described. The method, which is based on identification of the net analyte signal of single-beam spectra, can be calibrated using a single protein absorbance measurement and is thus well suited for crystallization monitoring where the quantity of protein is limited. The method is applied to measurements of glucose-isomerase concentration in a sodium phosphate buffer that is actively varied over the temperature range of 4-24 degrees C. The standard error of prediction using the optimized spectral range of 4670-4595 cm(-1) is 0.12 mg/mL with no systematic trend in the residuals with solution temperature. The method is also applied to previously collected spectra of hen egg-white lysozyme and yields a standard error of prediction of 0.14 mg/mL. Spectra sampled at discrete wavelengths can also be used for calibration and prediction with performance comparable to that obtained with spectral bands. A set of four wavelengths are identified that can be used to predict concentrations of both proteins with a standard error less than 0.14 mg/mL.

Static light scattering studies of OmpF porin: implications for integral membrane protein crystallization.

Integral membrane proteins carry out some of the most important functions of living cells, yet relatively few details are known about their structures. This is due, in large part, to the difficulties associated with preparing membrane protein crystals suitable for X-ray diffraction analysis. Mechanistic studies of membrane protein crystallization may provide insights that will aid in determining future membrane protein structures. Accordingly, the solution behavior of the bacterial outer membrane protein OmpF porin was studied by static light scattering under conditions favorable for crystal growth. The second osmotic virial coefficient (B22) was found to be a predictor of the crystallization behavior of porin, as has previously been found for soluble proteins. Both tetragonal and trigonal porin crystals were found to form only within a narrow window of B22 values located at approximately -0.5 to -2 X 10(-4) mol mL g(-2), which is similar to the "crystallization slot" observed for soluble proteins. The B22 behavior of protein-free detergent micelles proved very similar to that of porin-detergent complexes, suggesting that the detergent's contribution dominates the behavior of protein-detergent complexes under crystallizing conditions. This observation implies that, for any given detergent, it may be possible to construct membrane protein crystallization screens of general utility by manipulating the solution properties so as to drive detergent B22 values into the crystallization slot. Such screens would limit the screening effort to the detergent systems most likely to yield crystals, thereby minimizing protein requirements and improving productivity.

Partial least square analysis of lysozyme near-infrared spectra.

Proteins possess strong absorption features in the combination range (5000-4000 cm(-1)) of the near infrared (NIR) spectrum. These features can be used for quantitative analysis. Partial least squares (PLS) regression was used to analyze NIR spectra of lysozyme with the leave-one-out, full cross-validation method. A strategy for spectral range optimization with cross-validation PLS calibration was presented. A five-factor PLS model based on the spectral range between 4720 and 4540 cm(-1) provided the best calibration model for lysozyme in aqueous solutions. For 47 samples ranging from 0.01 to 10 mg/mL, the root mean square error of prediction was 0.076 mg/mL. This result was compared with values reported in the literature for protein measurements by NIR absorption spectroscopy in human serum and animal cell culture supernatants.

Temperature-independent near-infrared analysis of lysozyme aqueous solutions.

Digital Fourier filtering is used to produce a temperature-insensitive univariate calibration model for measuring lysozyme in aqueous solutions. Absorbance spectra over the 5000-4000 cm-1 spectral range are collected for lysozyme standards maintained at 14 degrees C. These spectra are used to compute the calibration model while a set of spectra collected at temperatures ranging from 4 to 24 degrees C are used to validate the accuracy of this model. The root-mean-square error of prediction (RMSEP) is 0.279 mg/mL over a tested lysozyme concentration range of 0.036-51.6 mg/mL. The detection limit is 0.68 mg/mL. In addition, multivariate calibration models based on partial least-squares regression (PLS) are evaluated and compared to the results from the univariate model. PLS outperforms the univariate model by providing a RMSEP of 0.090 mg/mL. Analysis of variance showed that both calibration methods effectively eliminate the adverse affects created by variations in solution temperature.

New strategies for protein crystal growth.

Protein crystallization is the most difficult and time-consuming step in the determination of a protein's atomic structure. As X-ray diffraction becomes a commonly available tool in structural biology, the necessity for rational methodologies and protocols to produce single, high-quality protein crystals has come to the forefront. The basics of protein crystallization conform to the classical understanding of crystallization of small molecules. Understanding the effect of solution variables such as pH, temperature, pressure, and ionicity on protein solubility allows the proper evaluation of the degree of supersaturation present in protein crystallization experiments. Physicochemical measurements such as laser light scattering, X-ray scattering, X-ray diffraction, and atomic force microscopy provide a clearer picture of protein crystal nucleation and growth. This ever deepening knowledge base is generating rational methods to produce protein crystals as well as means to improve the diffraction quality of such protein crystals. Yet, much remains unclear, and the protein crystallization research community will be quite active for many years to come.

Estimating lysozyme crystallization growth rates and solubility from isothermal microcalorimetry.

A microcalorimetric technique has been developed to measure crystal-growth kinetics and enthalpies of crystallization. The enthalpy of crystallization of hen egg-white lysozyme in 0.1 M acetate buffer at pH 4.6 was determined at 287 K using this technique. The enthalpies were directly measured to be -14.3 +/- 2.0 and -14.6 +/- 1.3 kcal mol-1 (1 kcal mol-1 = 4.184 kJ mol-1) for 3 and 5% NaCl solutions, respectively, which is in good agreement with values estimated from previous solubility measurements. Non-linear regression of the transient heat flow allowed measurement of the crystal growth rate as a function of protein supersaturation as well as the solubility. The crystal growth rates determined by this method were found to agree with those in the literature under the same solution conditions.

Stability of nicotinamide adenine dinucleotide immobilized to cyanogen bromide activated agarose.

The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose(R)-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d(-1), depending on solution pH. (c) 1997 John Wiley & Sons, Inc.

Surfactant structure effects in protein separations using nonionic microemulsions.

In this article, the extraction of cytochrome c utilizing various nonionic surfactant microemulsions has been tested to determine the effect of surfactant structure on protein partitioning. Surfactants tested include a linear alcohol ethoxylate (Neodol 91-2.5), two alkyl phenol ethoxylates (lgepal CO-520, Trycol 6985), and a series of alkyl sorbitan esters that are either ethoxylated (Tweens) or un-ethoxylated (Spans). Initial attempts to extract hemoglobin into Neodol 91-2.5 Winsor II microemulsions (oil-continuous) appeared successful based on heme estimation. Careful analysis showed that the hemoglobin had dissociated prior to extraction and that only the heme was extracted with false positive results. In fact, Neodol 91-2.5 microemulsions were unable to extract a variety of proteins with differing biophysical properties. Among all the other nonionic surfactant microemulsions tested only those made using sorbitan esters extracted significant amounts of cytochrome c. The partition coefficients achieved in this study are more than an order of magnitude higher than that seen previously in the literature for comparable sorbitan systems. However, this partition coefficient is extremely sensitive to ionic strength. At an ionic strength as low as 0.001 M, the partition coefficient is reduced to that seen in previous studies. We have found that protein partitioning in sorbitan ester microemulsions is not a function of water content. In addition, extraction is not a function of either alkyl chain length, or polyethylene oxide molecular weight. Hence, the sorbitan group appears to have an important role in extraction, possibly through a weak electrostatic protein-surfactant interaction. (c) 1995 John Wiley & Sons, Inc.

Evaluation of changes in skeletal muscle blood flow in the dog with contrast ultrasonography.

Intraoperative methods to assess skeletal muscle blood flow or muscle-flap perfusion during vascular reconstructive surgery are limited. At present, techniques enable only anatomic identification of the degree of patency of large vessels. We report here the first use of ultrasonography to assess dynamic changes in skeletal muscle perfusion. Baseline blood flow in the adductor muscle group of the hindlimbs of seven dogs was measured with an electromagnetic flow probe and with contrast ultrasound using the contrast agent Albunex. Blood flow was manipulated in each dog pharmacologically with random administration of intraarterial injections of Neo-Synephrine and papaverine. After each change in blood flow detected by electromagnetic flow probe, flow also was assessed qualitatively by four independent observers who graded video-recorded contrast enhancement in the muscle group on a 0 to 4 scale. Videodensitometry also was used to generate time versus intensity curves in the adductor muscle region of interest. Peak pixel intensity was determined during each flow condition. A total of 21 flow measurements were made with each assessment scheme (electromagnetic flow probe, video enhancement, videodensitometry) for each condition (7 control, 7 papaverine, 7 Neo-Synephrine). Changes in blood flow assessed by video enhancement scores and changes in peak pixel intensity correlated with changes measured by electromagnetic flow probe (r = 0.84 and 0.66, respectively). We conclude that contrast ultrasound may be used to detect changes in skeletal muscle perfusion intraoperatively. Measures of muscle perfused by visual inspection of contrast enhancement and videodensitometric data were in agreement with direct measurements of changes in skeletal muscle blood flow.

The relationship between immediate outcome after cardiac surgery, homogeneous cardioplegia delivery, and ejection fraction.

BACKGROUND: Optimal myocardial protection during cardiac surgery with ischemic arrest is predicated on among other variables, homogeneous cardioplegia distribution. Contrast echocardiography has been shown to provide information regarding the intramyocardial distribution of cardioplegia solution. To test the hypothesis that information regarding cardioplegia distribution derived from contrast echocardiography may be associated with immediate clinical outcome after cardiac surgery, data from 21 patients were examined retrospectively. METHODS: Contrast-enhanced cardioplegia distribution patterns of the left ventricle short axis view obtained with transesophageal echocardiography were examined off-line by four observers blinded to clinical outcome. Contrast effect was scored for eight equally divided myocardial segments (0 = no contrast, 1 = nonuniform contrast, 2 = uniform contrast, 3 = excessive contrast). The scores were then averaged between segments and between observers to generate an antegrade, a retrograde, and a combined global contrast score for each patient. RESULTS: Seventeen patients were separated from bypass without difficulty (group A) and 4 patients required sustained inotropic therapy or an intra-aortic balloon pump to facilitate separation from bypass (group B). As would be expected, group A patients had a higher average preoperative ejection fraction than did group B patients (60 percent +/- 14 vs 31 percent +/- 7, p < 0.01). In group A, however, for 4 of 17 patients (23 percent), low preoperative ejection fraction was not predictive of postoperative exogenous circulatory support requirements. Group A patients also had significantly higher antegrade (1.6 vs 1.2, p < 0.02), retrograde (1.7 vs 1.1, p < 0.02), and combined global contrast scores (1.7 vs 1.1, p < 0.01) than did group B patients. All patients with low preoperative ejection fraction and low intraoperative contrast scores required exogenous support to separate from cardiopulmonary bypass. CONCLUSION: Contrast echocardiography makes possible an evaluation of the intensity and distribution of contrast-enhanced cardioplegia delivery and we believe the efficacy of intraoperative myocardial protection. Although low preoperative ejection fraction is a known predictor of poor immediate postoperative outcome following cardiac surgery, not all patients with low preoperative ejection fractions require inotropic support postoperatively. Our results suggest that monitoring cardioplegia distribution with contrast echocardiography may offer insight for better patient stratification based on intraoperative myocardial protection in patients with low ejection fraction. We believe a more extensive evaluation of this relationship should be pursued in a prospective manner.

Intraoperative perfusion echocardiography.

The ability to assess perfusion intraoperatively should enable end-organ evaluation of the effects of therapeutic choices and provide a basis for understanding the mechanisms of disease. Several experimental techniques for assessment of tissue perfusion are being evaluated; contrast echocardiography appears to be adaptable to the perioperative setting because of its portability and relatively modest cost. With further improvements in commercial ultrasound imaging devices and ultrasonic contrast agents, intraoperative contrast echocardiography may prove to be a technique for quantitation of tissue perfusion. Contrast echocardiography is currently being used intraoperatively to assess cardioplegia distribution, coronary bypass graft patency, and coronary artery collateral vessel distribution. In addition, relative change in renal blood flow can be assessed during renal transplant surgery. With continued advancement of ultrasound technology providing linear (or known) acoustic signal response and wider dynamic range for detection of small and large concentrations of contrast agents, tissue blood flow may soon be evaluated with even greater precision.

Pitfalls in quantitative contrast echocardiography: the steps to quantitation of perfusion.

Current methods used clinically to assess myocardial perfusion are invasive and expensive. As the technology of ultrasound imaging improves, CE may provide a relatively inexpensive, noninvasive means of quantitating myocardial perfusion. Issues regarding stability of microbubble contrast agents must be studied more closely under physiologic conditions. As such, encapsulated microbubbles may provide more stability under physiologic pressures than free gas microbubbles. Introducing high concentrations of contrast, either by hyperconcentrating the contrast agent or by increasing the injection rate, may provide greater stability under physiologic conditions. Further, before quantitative statement of tissue perfusion can be made, the relationship between tracer concentration and system response must be established. Further, a "linear" postprocessing ultrasound setting does not eliminate this requirement as data must still undergo nonlinear transformation during log compression and time-gain compensation. Additionally, issues regarding "electronic thresholding" must be explored more extensively in vivo. Commercial ultrasound scanners, in their present form, may not offer adequate sensitivity for absolute quantitative studies. Further development of modified ultrasound systems may provide sufficient sensitivity for quantitative perfusion imaging. CE offers a potentially powerful tool in the clinical management of patients with ischemic heart disease. Conventional coronary angiography provides information on the size of a lesion, but accompanying tissue perfusion distal to the lesion cannot be determined. Doppler ultrasonography determines velocity of blood flow in large vessels but does not offer the potential to quantitate tissue perfusion. Clearly, CE has a place in the future of diagnostic imaging. The recent work of Ito et al. demonstrated the qualitative potential of CE in the identification of "areas at risk" in patients who had undergone thrombolysis or percutaneous transluminal coronary angioplasty after an acute myocardial infarction. With further improvement in the ultrasound imaging techniques and microbubble stability, CE may offer an inexpensive, noninvasive means of assessing myocardial perfusion.

Assessment of renal blood flow with contrast ultrasonography.

Sonicated albumin microspheres, a digitalizing ultrasound system, and a mathematical model for flow were used to determine whether blood flow in the canine kidney could be assessed with contrast ultrasound. Albunex ultrasound contrast microspheres were injected into the aorta while ultrasound images of the kidney and aorta were recorded simultaneously. Ultrasound data were obtained during contrast injections at 93 different renal blood flow rates in nine dogs. Contrast dose was calibrated to ultrasound system response for both aortic and renal images. A linear relationship between microbubble concentration used and pixel intensity was established (r = 0.89 for aortic images and r = 0.91 for renal images). Renal blood flow was manipulated from baseline by means of a hydraulic renal artery occluder and by intravenous dopamine or fenoldopam infusion. Blood flow calculated with contrast ultrasonography was compared with direct measurement obtained with an electromagnetic flow probe at each flow rate. Direct measurement correlated with rates calculated with contrast ultrasonography (r = 0.84, 95% confidence limits from 0.75 to 0.90). Overall, calculations tended to overestimate absolute flow measurements, and overestimation of flow tended to be greater during pharmacologically manipulated flow rates. We conclude the changes and trends in renal blood flow can be serially assessed in vivo with contrast ultrasonography, but technical limitations of present commercial ultrasounds systems preclude absolute quantification at this time.