Showing papers in "Methods in Enzymology in 1997"

Processing of X-ray diffraction data collected in oscillation mode

Abstract: Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.

Raster3D: photorealistic molecular graphics.

Abstract: Publisher Summary This chapter discusses Raster3D, which is a suite of programs for molecular graphics. Crystallographers were among the first and most avid consumers of graphics workstations. Rapid advances in computer hardware, and particularly in the power of specialized computer graphics boards, have led to successive generations of personal workstations with ever more impressive capabilities for interactive molecular graphics. For many years, it was standard practice in crystallography laboratories to prepare figures by photographing directly from the workstation screen. No matter how beautiful the image on the screen, however, this approach suffers from several intrinsic limitations. Among these is the inherent limitation imposed by the effective resolution of the screen. Use of the graphics hardware in a workstation to generate images for later presentation can also impose other limitations. Designers of workstation hardware must compromise the quality of rendered images to achieve rendering speeds high enough for useful interactive manipulation of three-dimensional objects.

SHELXL: high-resolution refinement.

Abstract: Publisher Summary SHELXL-93 was originally written as a replacement for the refinement part of the small-molecule program SHELX-76. The program is designed to be easy to use and general for all space groups and uses a conventional structure-factor calculation rather than a fast Fourier transform (FFT) summation. The latter would be faster but in practice involves some small approximations and is not suitable for the treatment of anomalous dispersion or anisotropic thermal motion. The price to pay for the extra precision and generality is that SHELXL is much slower than programs written specifically for macromolecules. This is compensated for, to some extent, by the better convergence properties, reducing the amount of manual intervention required. A new version, SHELXL-97, was released in May 1997; this is the version described in the chapter. The changes are primarily designed to make the program easier to use for macromolecules. Advances in cryogenic techniques, area detectors, and the use of synchrotron radiation enable macromolecular data to be collected to higher resolution than was previously possible. In practice, this tends to complicate the refinement because it is possible to resolve finer details of the structure. It is often necessary to model alternative conformations, and in a few cases, even anisotropic refinement is justified.

[27] Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods

Abstract: Publisher Summary This chapter discusses the maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) The chapter describes its extension to probability distributions incorporating anomalous diffraction effects, as well as measurement error and nonisomorphism Integrating these distributions in a whole complex plane leads to likelihood functions that can be used for heavy-atom detection and refinement and for producing phase-probability distributions The current implementation of this formalism in the computer program statistical heavy-atom refinement and phasing (SHARP) is also described in the chapter Likelihood functions can be used for the final phasing and calculation of Hendrickson–Lattman coefficients Numerical tests have been performed for three types of common refinements—namely, single isomorphous replacement, multiple isomorphous replacement with anomalous scattering (MIRAS), and MAD—and the results are summarized in the chapter A key feature of SHARP is its ability to refine lack-of-isomorphism parameters along with all the others

VERIFY3D: assessment of protein models with three-dimensional profiles.

Abstract: Publisher Summary The three-dimensional (3D) profile of a protein structure is a table computed from the atomic coordinates of the structure that can be used to score the compatibility of the 3D structure model with any amino acid sequence. Three-dimensional profiles computed from correct protein structures match their own sequences with high scores. An incorrectly modeled segment in an otherwise correct structure can be identified by examining the profile score in a moving-window scan. Thus, the correctness of a protein model can be verified by its 3D profile, regardless of whether the model has been derived by X-ray, nuclear magnetic resonance (NMR), or computational procedures. For this reason, 3D profiles are useful in the evaluation of undetermined protein models, based on low-resolution electron-density maps, on NMR spectra with inadequate distance constraints, or on computational procedures. An advantage of using 3D profiles for testing models is that profiles have not themselves been used in the determination of the structure. Traditional R-factor tests in X-ray analysis depend on the comparison of observed properties—that is, the X-ray structure factor magnitudes with the same property calculated from the final protein model.

[29] Preparation of selenomethionyl proteins for phase determination.

Abstract: Publisher Summary The use of selenomethionyl proteins for phase determination is growing in popularity for isomorphous replacement or multiwavelength anomalous dispersion (MAD) experiments. In some cases, it provides crucial phasing information and the key to solve a crystal structure. With the increase in the availability of synchrotron facilities (ESRF, APS) with beam lines dedicated to crystallographic studies, and MAD data collection in particular, selenomethionyl proteins may find routine use in phase determination. The procedures for engineering and crystallizing selenomethionyl proteins are divided into four steps: expression, cell growth, purification, and crystallization. The storage and properties of selenomethionyl protein crystals are discussed in this chapter. Selenomethionyl protein crystals should be kept in a reducing medium containing dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) and stored in an anaerobic chamber. Selenomethionyl protein crystals tend to be isomorphous with the native crystals, and selenomethionine incorporation does not usually alter diffraction limits. However, in some cases, selenomethionyl protein crystals are more radiation sensitive than their natural counterparts.

Electron-density map interpretation.

Abstract: Publisher Summary Any errors that occur in a crystallographic project usually will be found before publication. Often, if an error has been made, the project will stall and there will be no publication. Introducing a serious error in a model can be different. This chapter discusses the kinds of error that might be made and why these errors are made. It discusses some of the features of the crystallographic model-building program O. Real errors in models occur with frequencies that are, fortunately, inversely proportional to the seriousness of the error. Building a molecular model from electron density is a complicated process. During the interpretation of an electron-density map, the basic function of the molecular graphics program is to assist the scientist in imagining, and then remembering, the three-dimensional folding and features of the structure. Thus, it is important to be able to change the model quickly and not to be interrupted by the details of operating a computer program. To facilitate the rapid building and rebuilding of molecular models, O incorporates autobuild options, allowing the user to create a molecular structure quickly from a rough three-dimensional sketch. This has the drawback of possibly making it even easier to build a wrong structure.

Microbiological assay for serum, plasma, and red cell folate using cryopreserved, microtiter plate method.

Abstract: Publisher Summary This chapter presents microbiological assay for serum, plasma, and red cell folate, using cryopreserved, microtiter plate method. In the 500-ml beaker a solution of 0.5% (w/v) sodium ascorbate is prepared by dissolving 2.5 g of sodium ascorbate in 500 ml of water. This solution is used for preparing the working standard and for all assay dilutions. The working standard is prepared in the following way—a universal tube containing stock standard solution is brought to room temperature and a 50- μ l aliquot is taken and diluted to 100 ml with 0.5% (w/v) sodium ascorbate in a volumetric flask. This solution is mixed thoroughly, then 5 ml is taken and diluted to 100 ml with 0.5% (w/v) sodium ascorbate in a second volumetric flask to give a final working standard concentration of 500 ng/liter. Duplicate 50- μ l aliquots of serum or plasma or duplicate 25- μ l aliquots of whole-blood lysate are pipetted into labeled 4-ml polypropylene tubes. Using the adjustable repetitive sampling pipette the aliquots are diluted to a total of 1 ml with 0.5% (w/v) sodium ascorbate.

Antioxidant action of Ginkgo biloba extract EGb 761.

Abstract: Extracts from the leaves of Ginkgo biloba trees have been used therapeutically for centuries in traditional Chinese medicine, and in modern Chinese pharmacopoeias both the leaves and fruits are recommended for treating problems of heart and lungs. EGb 761 is a dry, powdered extract prepared from Ginkgo biloba leaves. It is a standardized mixture of several different chemical constituents; its two major classes of compounds are flavonoid glycosides and terpenoids (Drieu 1986; DeFeudis 1991). The flavonoid fraction is mainly composed of three flavonols: quercetin, kaempferol, and isorhamnetin, which are linked to a sugar(DeFeudis 1991). The terpenoid fraction is composed of ginkgolides and bilobalides(Drieu 1988). It also contains some organic acids, which help make it water-soluble(Drieu 1986).

[33] AMoRe: An automated molecular replacement program package

Abstract: Publisher Summary This chapter discusses the automated molecular replacement (AMoRe) program package. The basic problem of molecular replacement is to determine the positions of the molecules within a crystal cell. This is achieved by generating model crystal structures with molecular models placed at tentative positions and then selecting the configurations that give an acceptable agreement between the calculated structure factors and the observed ones. It constitutes a step toward the resolution of the crystal structure to be followed by model building and refinement procedures. The molecular replacement and a posteriori analysis of solved structures led to the optimization of the different parameters that define the rotation and translation functions to enhance the signal-to-noise ratio of the rotation function (RF) and translation function (TF) peaks. The success of the package, originally designed to deal with difficult problems, is a result of the combined action of its main characteristics: (1) many potential solutions, determined by revised or novel functions, are explored by means of fast algorithms, (2) correlation coefficients are used as the main criteria of selection, (3) the information coming from already positioned models is automatically incorporated into the procedure, and (4) there is a high degree of automation. The chapter describes the positional variables used in the package and discusses the strategy and results of AMoRe.

[28] Phase determination from multiwavelength anomalous diffraction measurements.

Abstract: Publisher Summary This chapter discusses phase determination from multiwavelength anomalous diffraction (MAD) measurements. The MAD approach to macromolecular structure determination has potential advantages for accuracy and convenience in phase evaluation. Isomorphism is intrinsically perfect; an algebraically exact analysis is possible; relative scattering strength and phasing power increase with scattering angle; and all required diffraction data can be measured from a single crystal. The virtual immortalization of crystals through freezing makes these advantages real and practical. Although many of the procedures in MAD phasing are in common with crystallographic practice, there are also a number of practical steps unique to this methodology. The chapter discusses the basic foundations of the method, presents the design and execution of experiments, and describes the steps and alternatives for data analysis.

[22] Detecting and overcoming crystal twinning

Abstract: Twinning is fairly common in protein crystals. In its merohedral from, twinning is not apparent in the diffraction pattern, but the observed intensities do not represent individual crystallographic intensities. Since partial twinning (twin fraction less than 1/2) and perfect twinning (twin fraction of 1/2) can both be identified relatively easily by examining intensity statistics, the appropriate tests should be performed routinely when working in space groups that support merohedral twinning.

[21] Data collection strategy

Abstract: Publisher Summary This chapter emphasizes the importance of determining the outer resolution limit to define the parameters to be used in data collection. The chapter describes the behavior of the reciprocal lattice during rotation photography, the effect of crystal mosaicity, the myth of the blind region, and the ways in which crystal symmetry can help. To design a data-collection protocol so that a data set is complete is not straightforward. Geometric considerations must be taken into account that depend on a crystal's orientation, its cell dimensions, and its symmetry. The geometric principles of the rotation method and individual cases of different symmetries of a crystal are discussed in the chapter. As the crystal is rotated during exposure of X-rays, the diffraction geometry may be explained by the Ewald-sphere construction, which, in turn, illustrates Bragg's law in three dimensions. The Ewald sphere with the radius 1/λ represents the radiation and therefore is stationary, whereas the crystal is represented by the reciprocal lattice with the origin at the point where the primary beam leaves the sphere. The crystal is rotated around one axis, usually perpendicular to the beam.

Detecting folding motifs and similarities in protein structures.

Abstract: Publisher Summary Detecting similarities at the level of tertiary structure is of interest for at least three reasons—namely, (1) it may provide insight into the modus operandi of proteins that share a common structural and functional trait, (2) it may reveal evolutionary pathways (either divergent or convergent), and (3) it may provide insight into protein folding and stability by revealing that a certain arrangement of helices and strands occurs in unrelated proteins. In all cases, if similarities at the tertiary structure level exist, sequence alignments based on these similarities are important. Such structure-based sequence alignments are expected to correlate with functional similarities. A cluster analysis using several known, high-resolution structures is carried out to find “typical” geometries of consecutive stretches of five Cα atoms in α helices and β strands, respectively.

Model building and refinement practice.

Abstract: Publisher Summary Model refinement has been a personalized affair for which laboratories have their preferred strategies, programs, etc. This has resulted in models with distinctive features of both the groups concerned and the software used. This chapter discusses the way a macromolecule should be refined and argues that the present practices in the community are often far from optimal, especially when only low-resolution data are available. All refinement programs nowadays use empirical restraints or constraints to ensure that a reasonable structure ensues during the refinement steps. This can result in a model with good stereochemical properties and also in a model in which molecules related by non-crystallographic symmetry (NCS) are forced to have similar (restrained) or identical (constrained) conformations. The aim of model building and refinement should be to construct a model that adequately explains the experimental observations, while making physical, chemical, and biological sense. It is a fact that low-resolution data can yield only low-resolution models. The refinement process, in particular, should always be tailored for each problem individually, keeping in mind the amount, resolution, and quality of the data.

Free R value: cross-validation in crystallography.

Abstract: Publisher Summary This chapter focuses on the free R value and other applications of cross-validation in crystallography. Powerful methods have been developed to lower the chances of misinterpreting or overinterpreting diffraction data. Some of the more important methods measure the agreement of the structure with empirical rules about protein folds, comprehensive conformational analyses, the real-space correlation coefficient, and the free R value. The protein-folding rules and conformational analyses depend on empirical knowledge of protein structure. They validate the model regardless of the fit to the diffraction data. In contrast, the real-space correlation coefficient and the free R value are entirely diffraction data based and are applicable to any macromolecule. They validate the extent to which the model explains the diffraction data. Even the best bulk solvent and multiconformer models result in free R values of around or slightly above 20% for the penicillopepsin crystal structure. A free R value of 20% is significantly higher than expected from the estimated high statistical quality of the diffraction data. Thus, it is conceivable that the present models for solvation and thermal motion are incomplete. Alternatively, the intensity data might be affected by systematic errors of unknown origin. These facts point to the need for the solution of benchmark macromolecular structures at high resolution and with accurate experimental phases.

Automated refinement for protein crystallography.

Abstract: Publisher Summary This chapter discusses an automated refinement procedure (ARP) for proteins. The basis of ARP is the iterative use of unrestrained least-squares minimization coupled with constant updating of the model. This is comparable to the iterative least-squares/Fourier synthesis approach for small molecules. It requires X-ray data to 2.0 A, or better, to allow unrestrained refinement and improvement of the whole content of the unit cell. At lower resolutions, as a rule, only unrestrained parts of the model are expected to be improved. The quality of data and the initial phase set greatly influences the power of ARP. Applied to the refinement of a medium-size structure at 1.0 A resolution and starting from one heavy-atom position, ARP determined the complete structure in a fully automated manner. ARP uses atomicity as the main property of the structure and differs completely from, for example, direct methods that are based on atomicity through statistical relationships between amplitudes of structure factors.

Standard Fmoc protocols

Abstract: Publisher Summary This chapter discusses standard Fmoc protocols. This chapter describes standard protocols for Fmoc solid-phase peptide synthesis. It discusses Fmoc-amino acid synthesis, compatible side-chain protection, solid phases (and the derivatization of), linkage agents, assembly methods, cleavage from the solid support, and workup and purification protocols. The method that emerged is operationally simple and chemically less complex than the Boc procedure. It is a mild procedure, and because of the Fmoc base lability and orthogonal nature relative to the previously accepted acid-labile protecting groups, it has allowed an element of chemical versatility in solid-phase strategies. It is the method of choice for the solid-phase synthesis of most modified peptide species including phosphorylated, sulfated, and glycosylated peptides. Its use in combination with groups such as Boc, allyl, and Dde has allowed access to a new dimension of peptide species from solid-phase synthesis procedures.

Lipases and alpha/beta hydrolase fold.

Abstract: Publisher Summary The three-dimensional structures of more than 20 representatives of the α/β hydrolase fold are now known and many more members have been identified by sequence and secondary structure comparisons. The fold is proving to be a common and stable way to assemble a wide variety of catalytic activities. With emphasis on lipases, this chapter reviews the features of this fold and the resources used to identify similarities in the rapidly growing number of enzymes and proteins that share this fold. The enzymes in this fold family include peroxidases, proteases, lipases, esterases, dehalogenases, and epoxide hydrolases. This fold is versatile in terms of the identities of catalytic residues and in their locations. The amino acids thus far observed as catalytic nucleophiles are serine, cysteine, and aspartate and both glutamate and aspartate have been observed as the catalytic acid. Although the acid is generally located after strand β7, functional triads can also be constructed with the acid located after strand β6. This fold family is also known to include proteins with no catalytic activity.

PHASES-95: a program package for processing and analyzing diffraction data from macromolecules.

Abstract: Publisher Summary The PHASES software package, including earlier releases, has been found to be both efficient and easy to use by many researchers and has been effective in the solution of a large number of protein structures. The basic philosophy employed while developing PHASES was to create software that is reasonably thorough, general, simple to use and understand, flexible, easy to interface with other software, and efficiently implemented on most popular hardware, particularly workstations. PHASES focus on solution of the phase problem in macromolecular crystallography, primarily by the methods of isomorphous replacement, anomalous scattering, solvent flattening, negative density truncation, phase extension, noncrystallographic symmetry averaging, and partial structure phase combination. Software tools are provided to start with unique reflections for native and/or derivative data sets and ultimately produce from them electron-density maps and skeletons that can be directly displayed in popular graphics programs for chain tracing. The package currently consists of 44 individual Fortran programs, about a dozen of which are “workhorse” programs used in most applications, and a single C interface subroutine used by some of the graphics programs.

Collaborative Computational Project, number 4: providing programs for protein crystallography.

Abstract: Publisher Summary Collaborative Computational Project, number 4 (CCP4) is funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) to support the development and use of computational tools in the field of macromolecular crystallography. Its first aim is to make available to the community a suite of programs to aid the determination of macromolecular structures by crystallography. Its second, and equally important, aim is to promote discussion on state-of-the-art techniques and to educate users in these techniques and the associated computer programs. The latter aim is pursued via an annual workshop, a twice-yearly newsletter, and an active e-mail discussion list. As part of the educational remit of CCP4, the annual Study Weekend takes place at the beginning of January. Each year, a particular topic is chosen, and a series of introductory talks and talks that are more specialized is given. These cover background theory, specific algorithms and programs, and instructive case studies. The resulting published proceedings are often one of the most up-to-date texts available on the chosen topic. The funding of CCP4, including important contributions from industrial companies, has grown to allow the employment of full-time staff based at Daresbury Laboratory to coordinate the activities of the project.

[34] Rotation function calculations with GLRF program

Abstract: Publisher Summary This chapter discusses the rotation function calculation with the GLRF program. The calculation of rotation functions represents the first step in structure determination by the molecular-replacement method. Rotation functions can be used to determine the orientation of a noncrystallographic axis in a crystal or the orientation of a search model relative to the molecules in a crystal. In the second step, rotational parameters can be used to determine the position of the noncrystallographic symmetry in the crystal or the position of the search model in the crystal. The information on the orientation and position of a search model leads to an initial solution for the crystal structure. Finally, the orientation and position of the noncrystallographic symmetry axes can be used to determine an initial phasing set, as well as to improve and extend the phase information.

Protein Data Bank archives of three-dimensional macromolecular structures.

Abstract: Publisher Summary This chapter discusses the Protein Data Bank (PDB) archives of three-dimensional (3D) macromolecular structures. Several pieces of information related to an entry are archived by the PDB. In addition to the coordinate entry file, the PDB stores files related to the experiment such as structure factors, nuclear Overhauser effect (NOE) restraints, and lists of chemical shifts. Also archived are auxiliary files used in structure analysis and refinement such as X-PLOR parameter and topology files. Currently, the archives are managed as a set of individual files, and each entry may have several associated files. The PDB is in the process of building a relational database, 3Dbase that will replace the current data management and access system. A description of 3DBase, including an outline of the way users can access its contents is provided in the chapter. Coordinate entries in the PDB are stored in separate files, each of which reports the results of an experiment or analysis that elucidates the structure of proteins, nucleic acids, polysaccharides, and other biological macromolecules. Although most of the data are generated from single crystal X-ray diffraction studies, a growing number of PDB entries are from nuclear magnetic resonance (NMR) studies.

Role of 1 alpha,25-dihydroxyvitamin D3 in osteoclast differentiation and function.

Abstract: Publisher Summary This chapter describes the methods for examining osteoclast development and function. Cytochemical staining for tartrate-resistant acid phosphatase (TRAP) is widely used for identifying osteoclasts in vivo and in vitro. Osteoclasts are shown to possess abundant calcitonin receptors. Expression of calcitonin receptors is one of the most reliable markers for identifying osteoclasts. For autoradiography of 125 I-labeled calcitonin, cultures are performed on plastic coverslips placed in 24-well culture plates. Cocultures of primary osteoblastic cells with bone marrow cells or spleen cells produce more osteoclasts than bone marrow cultures do. Osteoclasts formed on plastic culture dishes cannot be detached by treatment with either trypsin-EDTA or bacterial collagenase. To obtain functionally active osteoclasts formed in cocultures, a collagen gel culture is developed. Osteoclasts adhere to the bone surface through specialized discrete structures called podosomes in the clear zone, which consist mainly of dots containing F-actin. Therefore, the ringed structure of podosomes (actin ring) formed in osteoclasts is a characteristic of polarized osteoclasts. The actin rings are visualized by staining F-actin with rhodamine-conjugated phalloidin.

[6] Second virial coefficient as predictor in protein crystal growth.

Abstract: Publisher Summary Structure determination of a protein depends on the availability of the crystals of the protein for X-ray diffraction analysis. Numerous physical and chemical factors influence the outcome of protein-crystallization experiments. Screening methods generally rely on varying protein concentration, solution pH, or crystallizing agent type and concentration. To reduce the number of screening experiments necessary to find and optimize crystal-growth conditions, sampling methods that can increase the efficiency of crystal-growth screening have been proposed. These advanced screening methods, perhaps coupled with robotic systems capable of automatically setting up and evaluating hundreds of screening experiments, may shorten the path to successful protein crystallization. Evidence suggests that a dilute solution parameter called the osmotic second viral coefficient, B22, determined by static light scattering (SLS), represents an appropriate assay for predicting protein crystallization. The SLS results presented in the chapter give an unambiguous measure of B22. The analytical method that was used to study protein solutions was SLS utilizing a DAWNF laser photometer from Wyatt Technology; this method relied on measuring the intensity of light scattered by a protein solution as a function of the protein concentration.

Determination of vitamin K compounds in plasma or serum by high-performance liquid chromatography using postcolumn chemical reduction and fluorimetric detection.

Abstract: Publisher Summary The high-performance liquid chromatography (HPLC) assays used for the determination of endogenous phylloquinone, phylloquinone 2,3-epoxide, menaquinones, and most recently 2',3'-dihydrophylloquinone have evolved from the postcolumn reduction and fluorimetric detection methodology. The analytical system utilized to reduce vitamin K compounds to their fluorescent hydroquinones consists of a postcolumn, dry chemical reactor containing zinc metal. Hydroquinones are produced by chemical reduction over zinc in the presence of zinc ions, which are provided by the mobile phase. This on-line reduction process forms the core of the chromatographic systems used for the determination of vitamin K. Although the column configuration, injector type, mobile-phase composition, and flow rates of analytical systems may vary, the online, postcolumn reduction is essentially the same. This chapter describes two assays based on postcolumn reduction, but with significant enhancements made in the sensitivity of analytical systems: (1) a simplified method for the determination of fasting plasma or serum concentrations of phylloquinone and (2) an assay for the simultaneous determination of endogenous phylloquinone and phylloquinone 2,3-epoxide in plasma or serum.

A critical reevaluation of the phenomenon of interfacial activation

Abstract: Publisher Summary Lipases are carboxylic ester hydrolases and have been termed glycerolester-hydrolase in the international system of classification. They greatly differ with respect to both of their origins and their kinetic properties. They can catalyze in vitro the hydrolysis, or synthesis, of a wide range of different carboxylic esters; however, they all show a higher specific activity toward glyceridic substrates. Under physiological conditions, because natural triacylglycerols are water insoluble, lipases that are generally soluble in water, catalyze the hydrolysis of carboxylic ester bonds at lipid/water interfaces. Some lipases, such as gastric lipases, rapidly become denatured at an interface with a pure tributyrin or tributanoylglycerol emulsion. Consequently, it is impossible to assess experimentally what interfacial activation may have occurred. The three-dimensional structures of lipases suggested that the interfacial activation phenomenon might be due to the presence of an amphiphilic surface loop covering the active site of the enzyme in solution, just like a lid. When contact occurs with a lipid/water interface, this lid might undergo a conformational rearrangement as the result of which the active site becomes accessible.