4
TIBTECH - JANUARY 1991 [Vol. 9]
I 11 New attempts to solve protein structures lead to MADness Genome projects, by application of recombinant technology, are giving ready access to proteins both of known and unknown function. Will determination of their crystal structures ever be as routine? Crystallographer Wayne Hendrickson (Howard Hughes Medical Institute, Columbia, NY, USA), student John Horton and molecular biologist David LeMaster (Northwestern) are betting that it will and their answer to the problem is MAD 1. One could liken the process of solving a protein structure using Xray diffraction to scaling a Himalayan peak. In both cases the scientist (generally a graduate student!) faces a long climb. Since the heroic first ascents (myoglobin, haemoglobin, lysozyme), the strategy and hardware needed to ascend the lower slopes has improved. Obtaining the protein has become easier; expression of foreign structural genes in microorganisms has led to the production of numerous functional proteins in high yield. Crystallization remains an 'art', but progress has been made here too: recombinant proteins are frequently of high quality and determination of crystallization conditions has been automated. Unfortunately, many would-be expeditions are still defeated by the crux of the ascent - the 'phase p r o b l e m ' - and have to retreat to base. Isomorphism To continue the mountaineering analogy, the solution of this phase problem is somewhat like climbing the summit rocks blind. The molecular ascentionist can feel the rock (the amplitudes of the diffracted X-rays), but cannot determine the positions of the hand-holds (the relative phases of the diffracted Xrays). Both factors are essential to solve the structure. Progress is slow and involves much trial and error. The traditional technique is to dif~) 1991, Elsevier Science Publishers Ltd (UK)
fuse a heavy-metal compound into the protein crystal. Attachment to a specific site in each protein molecule, without otherwise altering the protein structure, modifies the X-ray intensities sufficiently for a difference to be measured and the relatively simple spatial arrangement of the heavy atoms to be deduced. Once such an isomorphous derivative is found, both phase and amplitude information for the heavy metal's contribution to the diffracted X-rays can be calculated. In general, this reduces the phase problem to a choice of two possibilities for each measured intensity (see Fig. la). A second isomorphous heavy-metal derivative, occupying a different site on the protein, then solves the phase problem. Unfortunately, the discovery of suitable derivatives is a trial-and-error process - many potential derivatives perturb the protein structure, and therefore do not meet the condition of isomorphism. Anomalous diffraction A powerful and potentially general solution to the phase problem arises when a protein contains a small number of heavy atoms which have an X-ray absorption at a wavelength just a little longer than that of the incident radiation. Under these conditions, anomalous diffraction occurs and the phase of diffracted X rays increases above normal. Anomalous X-ray diffraction has historical importance 2 because it was used to work out the first absolute configuration of an optically active chemical (rubidium tartrate), 100 years after Pasteur recognized that tartrates could exist in left- and right-handed forms. MADness Irradiation of the heavy atom in the protein crystal (Fig. 1) at a wavelength near its absorption edge leads
0167 - 9430/90/$2.00
to the phase and amplitude change shown in Fig. lb. Since this can also be calculated from the known positions of the heavy atoms, the additional information resolves the ambiguity without need for a second isomorphous derivative. If, instead of diffusing in the heavy atom, it is already part of the crystal structure, then irradiation at several defined wavelengths (such as is possible using synchrotron radiation), allows the determination of the spatial location of the heavy atoms as before, but this time the isomorphism is perfect. The differences in diffracted X-ray intensities determined at three or more different wavelengths then permit the solution of the phase problem in a similar way to that shown by the geometric constructions of Fig. 1. This way led to MADness (multi-wavelength anomalous diffraction). Selenium vs. the heavy metals By taking advantage of the ability of the protein-synthesizing machinery of E. coil to use selenomethionine instead of methionine, this analogue can be inserted into proteins. Although selenium, with just 18 electrons more than sulphur, is not in the same league as the usual heavy metals used (i.e. mercury and uranium), its anomalous scattering is nearly ideal for the MAD technique. Hendrickson and colleagues produced both phage T4 and E. coil thioredoxins containing selenomethionine using bacteria auxotrophic for methionine to ensure 100% incorporation of the amino acid analogue. Both thioredoxin structures were already known and the selenomethionyl versions crystallized essentially isomorphously. For the technique to be of general value, it was obviously important that the normal and selenomethionyl versions turned out to be isostructural. This is the case, since the Columbia group have now solved the structure of ribonuclease H by MAD analysis of the selenomethionyl protein ~. Despite the success so far, Hendrickson is cautious about the generality of the method. Clearly, proteins must contain some, but not too much, methionine (it occurs with an average frequency of I in 60 amino acids); selenomethionine is more easily oxidized than methionine. Although selenomethionine has been
TIBTECH - JANUARY 1991 [Vol. 9]
--Fig. 1
incorporated into soluble CD4 protein in Chinese hamster ovary cells 4, higher eukaryotic cells are more sensitive to the unnatural amino acid. However, it is clear that if a protein is already cloned and expressed in E. coli, going MAD might just get you that first ascent.
Heavy-atom solution of the phase problem. (a) Geometric representation of a single diffracted X ray from a protein crystal The red circle represents the unknown phase angle (0 to 360 degrees). The radius, Fp, is determined from the square root of the X-ray intensity measurement. Location of a heavy atom diffused into the protein crystal allows calculation of the ampfitude and of the phase of its contribution to the X-ray intensity. This is represented by the vector Fh. The heavy atom changes the diffracted X-ray intensity such that the amplitude is now Fph. Drawing a second circle (black outline) of this diameter, centred at the end of Fh, it can be seen that this cuts the first circle at two points. These are the two choices of phase angle. (b) is the same diffracted X ray as in (a), but now the X rays are close to the absorption edge of the heavy atom h. An additional (anomalous) diffraction occurs which advances the phase angle of Fh and changes its amplitude (Fh + Fh "). This can be calculated as before. Now a third (thicker) circle can be drawn with radius Fph" and this determines the correct choice of phase. In MAD, the heavy atom h is already part of the structure, so that additional wavelengths are used to provide the information obtained in (a) by the isomorphous derivative.
The proof of the cloning is in the eating In 1991, there will be a large number of applications to food-regulatory bodies [e.g. Food and Drugs Administration {FDA) in the USA, and the Ministry of Agriculture, Fisheries and Food (MAFF), in the UK], for approval of genetically engineered living material as food for human consumption. It will be a concerted onslaught, involving microorganisms, plants and higher animals, which will keep the special committees (Government Advisory Committee on Novel Foods, Advisory Committee for Genetic Manipulation) very busy indeed. How will the regulators act and the public react?
ranged so that a change in the animals' diet would greatly increase circulating levels of the hormone. Unlike the famous 'giant' mice z, the pigs did not show dramatic size increases, but instead had very lean muscle tissue - a desirable commercial feature. Unfortunately, the constitutively high levels of growth hormone led to diabetes, sterility and other pathologies. Insufficient attention had been paid to the endocrinologists 1, since release of growth hormone is known to be periodic and not constitutive. However, the potential for modifying livestock was established.
Livestock modification The tip of the iceberg was exposed in 1988-1989. In the USA, thousands of domestic pig embryos were injected with transgenes encoding growth hormones (somatotropins) a. The promoters for these were ar-
Modification of plants, yeast and fish In the world of plants, ICI has produced transgenic tomatoes which express an antisense mRNA, designed to reduce the amount of a cell-wall degrading enzyme. Such tomatoes can be stored, without
~) 1991, Elsevier Science Publishers Ltd (UK) 0167- 9430FJ0/$2.00
References 1 Hendrickson, W. A., Horton, J. R. and LeMaster, D.M. (1990) EMBO J. 9, 1665-1672 2 Bockhoven, C., Schoone, J. C. and Bijvoet, J, M. (1951) Acta Crystallogr. 4, 275-284 3 Yang, W., Hendrickson, W. A., Crouch, R. J. and Satow, Y. (1990) Science 249, 1398-1405 4 Deem K. C., McDougal, J. S., Inacker, R. Folena-Wasserman, G., Arthos, J., Rosenberg, J., Madden, P.J., Axel, R. and Sweet, R.W. (1988) Nature 331, 82-84 MICHAEL GEISOW
Biodigm, 115 Main Street, East Bridgeford, Nottingham NG13 8NH, UK.
softening, for prolonged periods. Meanwhile, in the Netherlands, the Dutch yeast company Gist-Brocades gained approval for the use of a genetically engineered yeast in human food. Expression of the maltose-transport and -utilizing gone products was increased by means of a more efficient transcriptional promoter from the same strain of yeast. More carbon dioxide is produced, which decreases the rising time of the dough. It is also worth noting that the same company has been selling recombinant chvmosin {rennin}, for cheese produciion in Europe for two years, and expects FDA approval for sale in the USA shortly. The natural source of chymosin is the cow stomach. As described in a recent TIBTECH review 3, progress has been made with producing transgenic fish, incorporating commercially important features such as improved resistance to disease and to cold. Such new species will certainly be on the regulators' agendas, and possibly soon on their menus. The science which has led to this transgenic cornucopia represents an
TIBTECH - JANUARY 1991 [Vol. 9]
I 11 New attempts to solve protein structures lead to MADness Genome projects, by application of recombinant technology, are giving ready access to proteins both of known and unknown function. Will determination of their crystal structures ever be as routine? Crystallographer Wayne Hendrickson (Howard Hughes Medical Institute, Columbia, NY, USA), student John Horton and molecular biologist David LeMaster (Northwestern) are betting that it will and their answer to the problem is MAD 1. One could liken the process of solving a protein structure using Xray diffraction to scaling a Himalayan peak. In both cases the scientist (generally a graduate student!) faces a long climb. Since the heroic first ascents (myoglobin, haemoglobin, lysozyme), the strategy and hardware needed to ascend the lower slopes has improved. Obtaining the protein has become easier; expression of foreign structural genes in microorganisms has led to the production of numerous functional proteins in high yield. Crystallization remains an 'art', but progress has been made here too: recombinant proteins are frequently of high quality and determination of crystallization conditions has been automated. Unfortunately, many would-be expeditions are still defeated by the crux of the ascent - the 'phase p r o b l e m ' - and have to retreat to base. Isomorphism To continue the mountaineering analogy, the solution of this phase problem is somewhat like climbing the summit rocks blind. The molecular ascentionist can feel the rock (the amplitudes of the diffracted X-rays), but cannot determine the positions of the hand-holds (the relative phases of the diffracted Xrays). Both factors are essential to solve the structure. Progress is slow and involves much trial and error. The traditional technique is to dif~) 1991, Elsevier Science Publishers Ltd (UK)
fuse a heavy-metal compound into the protein crystal. Attachment to a specific site in each protein molecule, without otherwise altering the protein structure, modifies the X-ray intensities sufficiently for a difference to be measured and the relatively simple spatial arrangement of the heavy atoms to be deduced. Once such an isomorphous derivative is found, both phase and amplitude information for the heavy metal's contribution to the diffracted X-rays can be calculated. In general, this reduces the phase problem to a choice of two possibilities for each measured intensity (see Fig. la). A second isomorphous heavy-metal derivative, occupying a different site on the protein, then solves the phase problem. Unfortunately, the discovery of suitable derivatives is a trial-and-error process - many potential derivatives perturb the protein structure, and therefore do not meet the condition of isomorphism. Anomalous diffraction A powerful and potentially general solution to the phase problem arises when a protein contains a small number of heavy atoms which have an X-ray absorption at a wavelength just a little longer than that of the incident radiation. Under these conditions, anomalous diffraction occurs and the phase of diffracted X rays increases above normal. Anomalous X-ray diffraction has historical importance 2 because it was used to work out the first absolute configuration of an optically active chemical (rubidium tartrate), 100 years after Pasteur recognized that tartrates could exist in left- and right-handed forms. MADness Irradiation of the heavy atom in the protein crystal (Fig. 1) at a wavelength near its absorption edge leads
0167 - 9430/90/$2.00
to the phase and amplitude change shown in Fig. lb. Since this can also be calculated from the known positions of the heavy atoms, the additional information resolves the ambiguity without need for a second isomorphous derivative. If, instead of diffusing in the heavy atom, it is already part of the crystal structure, then irradiation at several defined wavelengths (such as is possible using synchrotron radiation), allows the determination of the spatial location of the heavy atoms as before, but this time the isomorphism is perfect. The differences in diffracted X-ray intensities determined at three or more different wavelengths then permit the solution of the phase problem in a similar way to that shown by the geometric constructions of Fig. 1. This way led to MADness (multi-wavelength anomalous diffraction). Selenium vs. the heavy metals By taking advantage of the ability of the protein-synthesizing machinery of E. coil to use selenomethionine instead of methionine, this analogue can be inserted into proteins. Although selenium, with just 18 electrons more than sulphur, is not in the same league as the usual heavy metals used (i.e. mercury and uranium), its anomalous scattering is nearly ideal for the MAD technique. Hendrickson and colleagues produced both phage T4 and E. coil thioredoxins containing selenomethionine using bacteria auxotrophic for methionine to ensure 100% incorporation of the amino acid analogue. Both thioredoxin structures were already known and the selenomethionyl versions crystallized essentially isomorphously. For the technique to be of general value, it was obviously important that the normal and selenomethionyl versions turned out to be isostructural. This is the case, since the Columbia group have now solved the structure of ribonuclease H by MAD analysis of the selenomethionyl protein ~. Despite the success so far, Hendrickson is cautious about the generality of the method. Clearly, proteins must contain some, but not too much, methionine (it occurs with an average frequency of I in 60 amino acids); selenomethionine is more easily oxidized than methionine. Although selenomethionine has been
TIBTECH - JANUARY 1991 [Vol. 9]
--Fig. 1
incorporated into soluble CD4 protein in Chinese hamster ovary cells 4, higher eukaryotic cells are more sensitive to the unnatural amino acid. However, it is clear that if a protein is already cloned and expressed in E. coli, going MAD might just get you that first ascent.
Heavy-atom solution of the phase problem. (a) Geometric representation of a single diffracted X ray from a protein crystal The red circle represents the unknown phase angle (0 to 360 degrees). The radius, Fp, is determined from the square root of the X-ray intensity measurement. Location of a heavy atom diffused into the protein crystal allows calculation of the ampfitude and of the phase of its contribution to the X-ray intensity. This is represented by the vector Fh. The heavy atom changes the diffracted X-ray intensity such that the amplitude is now Fph. Drawing a second circle (black outline) of this diameter, centred at the end of Fh, it can be seen that this cuts the first circle at two points. These are the two choices of phase angle. (b) is the same diffracted X ray as in (a), but now the X rays are close to the absorption edge of the heavy atom h. An additional (anomalous) diffraction occurs which advances the phase angle of Fh and changes its amplitude (Fh + Fh "). This can be calculated as before. Now a third (thicker) circle can be drawn with radius Fph" and this determines the correct choice of phase. In MAD, the heavy atom h is already part of the structure, so that additional wavelengths are used to provide the information obtained in (a) by the isomorphous derivative.
The proof of the cloning is in the eating In 1991, there will be a large number of applications to food-regulatory bodies [e.g. Food and Drugs Administration {FDA) in the USA, and the Ministry of Agriculture, Fisheries and Food (MAFF), in the UK], for approval of genetically engineered living material as food for human consumption. It will be a concerted onslaught, involving microorganisms, plants and higher animals, which will keep the special committees (Government Advisory Committee on Novel Foods, Advisory Committee for Genetic Manipulation) very busy indeed. How will the regulators act and the public react?
ranged so that a change in the animals' diet would greatly increase circulating levels of the hormone. Unlike the famous 'giant' mice z, the pigs did not show dramatic size increases, but instead had very lean muscle tissue - a desirable commercial feature. Unfortunately, the constitutively high levels of growth hormone led to diabetes, sterility and other pathologies. Insufficient attention had been paid to the endocrinologists 1, since release of growth hormone is known to be periodic and not constitutive. However, the potential for modifying livestock was established.
Livestock modification The tip of the iceberg was exposed in 1988-1989. In the USA, thousands of domestic pig embryos were injected with transgenes encoding growth hormones (somatotropins) a. The promoters for these were ar-
Modification of plants, yeast and fish In the world of plants, ICI has produced transgenic tomatoes which express an antisense mRNA, designed to reduce the amount of a cell-wall degrading enzyme. Such tomatoes can be stored, without
~) 1991, Elsevier Science Publishers Ltd (UK) 0167- 9430FJ0/$2.00
References 1 Hendrickson, W. A., Horton, J. R. and LeMaster, D.M. (1990) EMBO J. 9, 1665-1672 2 Bockhoven, C., Schoone, J. C. and Bijvoet, J, M. (1951) Acta Crystallogr. 4, 275-284 3 Yang, W., Hendrickson, W. A., Crouch, R. J. and Satow, Y. (1990) Science 249, 1398-1405 4 Deem K. C., McDougal, J. S., Inacker, R. Folena-Wasserman, G., Arthos, J., Rosenberg, J., Madden, P.J., Axel, R. and Sweet, R.W. (1988) Nature 331, 82-84 MICHAEL GEISOW
Biodigm, 115 Main Street, East Bridgeford, Nottingham NG13 8NH, UK.
softening, for prolonged periods. Meanwhile, in the Netherlands, the Dutch yeast company Gist-Brocades gained approval for the use of a genetically engineered yeast in human food. Expression of the maltose-transport and -utilizing gone products was increased by means of a more efficient transcriptional promoter from the same strain of yeast. More carbon dioxide is produced, which decreases the rising time of the dough. It is also worth noting that the same company has been selling recombinant chvmosin {rennin}, for cheese produciion in Europe for two years, and expects FDA approval for sale in the USA shortly. The natural source of chymosin is the cow stomach. As described in a recent TIBTECH review 3, progress has been made with producing transgenic fish, incorporating commercially important features such as improved resistance to disease and to cold. Such new species will certainly be on the regulators' agendas, and possibly soon on their menus. The science which has led to this transgenic cornucopia represents an
