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Repacking protein interiors Warren S. Sandberg and Thomas C. Terwilliger Several goals of protein engineering may be achieved through redesign and repacking of protein interiors. The effects of interior apolar substitutions on protein stability depend strongly on the site of the substitution. One reason for this is that protein interiors have properties both of apolar liquids and of crystalline solids. Substitutions at interior sites affect the stability of a protein by changing the hydrophobicity, but each site in a protein has a characteristic energy associated with introducing packing changes, and the net stability depends on both of these factors. Reconstructing protein interiors or designing them de novo is a major and challenging step in constructing new and altered proteins. The hydrophobic cores of naturally occurring proteins consist largely of tightly packed, interdigitating side chains whose arrangement defines the relationships between larger units of secondary and tertiary structure 1-7. In effect, interior packing determines much of the overall shape of a protein as well as the arrangements of the residues on its surface, some of which may be critical for function 8'9. By redesigning protein interiors, it may be possible to further stabilize existing proteins by optimizing the arrangements in their interiors, thereby reducing their susceptibility to extremes of temperature, pH or ionic conditions 1°'11. It may also be possible to alter active sites 12 or reposition protein surfaces. For example, internal packing differences in various globins 8 allow the relative positions of the B and E helices to differ by as much as 7 A. Finally, understanding how to pack protein interiors will be essential in the de novo design of proteins with novel properties 13-~5.

The hydrophobic effect The first approach emphasizes the hydrophobicity of protein interiors, where most side chains are relatively non-polar. Clustering of apolar atoms in hydrophobic cores stabilizes the folded forms of proteins through the hydrophobic effect 16n7. Models of the hydrophobic effect based on the properties of apolar liquids 18-2° suggest that it might be possible to predict the influence of core amino acids on protein stability using a liquid-like model of the protein interior. It is not thought that protein interiors are actually fluid, but rather that the detailed arrangement of interior side chains might not be a critical determinant of stability. The density of packing The alternative approach takes into account that protein interiors are very densely packed 5'6 and that the polypeptide chain of the protein ties the side chains within the core together. In addition, the corres-

ponding side chains in each molecule of a given protein usually have nearly identical conformations in protein crystals. This leads to a model suggesting that proteins are built around rigidly packed, nondeformable cores of apolar side chains. With this model, it may be possible to predict which amino acid side chains are physically permissible at a particular site in the interior of a protein, on the basis of the shape of the site and the shapes (and distinct conformations) of the amino acids 9. In practice, it seems certain that a combination of these models would be more useful than either one alone, and we propose a unified view of the effects of interior amino acid ~ubstitutions on protein stability that incorporates elements of both models.

Effects of interior amino acid substitutions on protein stability Analysis of the ability of internal sites to tolerate substitutions reveals two important principles, reflecting the two views of protein interiors. (1) Interior residues, with few exceptions, cannot be strongly polar, consistent with the interior compositions of natural proteins 21. (2) Combinations of interior side chains leading to deviations in core volume of more than a few methylene groups from the wild type do not lead to functional proteins 22. Four amino acid side chains commonly found buried inside proteins are the aliphatic side chains of Ala, Val, Ile and Leu (Fig. 1), which, along with the side chains of Cys, Met, Phe and Trp, are generally regarded as apolar. As saturated hydrocarbons, the aliphatic amino

--Fig. 1

Modeling protein interiors Two approaches have been used to model protein interiors, focusing on different properties of protein cores. Alanine (Ala) W. S. Sandberg and T. C. Terwilliger are at the Department of Biochemistry and Molecular Biology, The University of Chicago, 920 E. 58th Street, Chicago, IL 6O637, USA.

Valine (Val)

Isoleucine (tie)

Leucine (Leu)

Ball and stick models of four aliphatic amino acid side chains. The ball represents CHn (n= 1, 2, 3).

© 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91/$2.00

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T I B T E C H - FEBRUARY 1991 [Vol. 9]

--Fig. 2 a

acid side chains are all quite hydrophobic TM, and should contribute strongly to stability. Protein stability is generally measured by monitoring indicators of native structure as a function of denaturant concentration (frequently urea or guanidine hydrochloride), or of temperature. Decreased stability is indicated by loss of structure at lower denaturant concentration or temperature. The effects of interior aliphatic amino acid substitutions on protein stability (free energy changes upon unfolding, AGu), have been measured in detail in several cases, with widely differing results. For substitutions incorporating buried aliphatic residues at sites in tryptophan synthase (Glu49), barnase (Ile88, Ile96), and one site in the bacteriophage fl gene V protein (gVp) (Ile47), changes in stability are strongly correlated with changes in hydrophobicity, but with different proportionality constants 23-25 (i.e. the proportional change in stability relative to hydrophobicity will vary from one protein to another, and between sites in the same protein) (Fig. 2a). In contrast, for a site (Va175), in dihydrofolate reductase (DHFR), two sites (Ile3, Ala129) in T4 lysozyme, and a second site (Va135) in the gVp, the dependence of stability on side chain hydrophobicity is much smaller 23'26-28 (Fig. 2b). In some instances, aliphatic side chains, larger and presumably more hydrophobic than the wildtype residues, actually decreased

b

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Side chain hydrophobicity (kcal mo1-1) Stability of proteins containing interior aliphatic substitutions compared with the hydrophobicities of the substituting residues. (a) Substitution sites for which protein stability increases linearly with residue hydrophobicity: symbols; (x), site 49 of tryptophan synthase; (D), site 96 of barnase; ("), site 88 of barnase; (e), site 47 of gene V protein (gVp). (b) Sites in which protein stability is less dependent on side chain hydrophobicity: symbols; (+), site 75 of dihydrofolate reductase (DHFR); (o), site 35 of gVp; (A), site 3 of 7"4 Iysozyme; ( A ), site 129 of T4 lysozyme. (Stabilities are reported as AGu, the free energies of unfolding 23-25"28,except for the data for T4 lysozyme, which is reported as the change in free energy of unfolding relative to the wild-type protein 2e'27. Stabilities measured under the different conditions employed in the various studies are presumed to be comparable without introducing serious errors24"32.) Residue hydrophobicities, z~Gtr,cyclohexane--*water, are from Ref. 18.

responses to substitution within the same protein, as in the cases of gVp, barnase and T4 lysozyme, as well as the different responses between proteins to identical substitutions, suggest that the characteristics of protein interiors vary considerably from site to site 23-29.

stability 23,27,28.

Another way to look at this data is to compare the stability of each protein to the stability of a 'minimal' reference protein containing Ala (a small, aliphatic amino acid) at the site of substitution. Viewed this way, the stability of each protein varies in a fairly linear fashion with hydrophobicity when Ala is replaced by larger aliphatic residues, but, once again, with different constants of proportionality (Fig. 3). Recently, it has been demonstrated that the arrangement of core side chains, and not just the composition of the protein interior, is quite important in determining protein stability 29. This suggests that no simple relationship exists between the hydrophobicity of interior residues and stability. Taken together, the observations of site-dependent

U

A characteristic 'packing energy' for introducing large residues at each interior site One way to unify these results is to begin with the liquid model for a protein interior and then add features of the rigid model. The liquid model predicts that replacing small aliphatic side chains with large ones will stabilize the folded structure of a protein, increasing AGu. In this model, the contribution of side chain X to AGu is given by the free energy of transfer (AGtr.x) required to move X from an apolar solvent (representing the protein interior), to water (the environment of X in the unfolded protein). The stability contributions of two aliphatic side chains, Ala and Leu, for example, are given by AGtr,Ala and AGtr.Leu, respectively, and the differ-

ence in free energy change upon unfolding when Ala is replaced by Leu in a protein (AAGu.Ala-,Leu) is given by: AAGu,Ala---.Leu = AGtr,Leu -AGtr,Ala

(1)

Values of the free energies of transfer depend on the liquid hydrocarbon chosen as a reference phase. Cyclohexane is a useful reference phase because it is the least polar of the commonly used liquid models, and therefore likely to be the best gauge of purely hydrophobic effects 18. Now we add an element of the rigid model of the interior, suggesting that there is an energetic penalty (or benefit) for introducing a large aliphatic side chain at an interior site previously occupied by Ala, and that this parameter is characteristic of each site and proportional to the volume of the side chain. This 'packing energy' (AAGpacking) is the flee energy difference between placing a large side chain in the interior of a protein and placing it in a liquid hydrocarbon 29. The stability change

TIBTECH- FEBRUARY 1991 [Vol. 9]

61

--Fig. 3, --Fig. 4,

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Protein stability versus residue hydrophobicity with stability and hydrophobicity expressed relative to the side chain of Ala. Stabilities of seven of the substituted proteins from Fig. I are shown, but for each site stabilities are plotted as the difference between the stability of the mutant and a reference protein containing Ala at the site of interest. Similarly, hydrophobicities are shown as the increase in hydrophobicity of each side chain relative to that of alanine. Symbols, as for Fig. 2; (×), site 49 of tryptophan synthase; (D), site 96 of barnase; ("), site 88 of barnase; (e), site 47 of gVp; (+), site 75 of DHFR; (o), site 35 of gVp; (A), site 3 of 7"4 lysozyme.

accompanying the replacement of Ale by Leu in a protein interior becomes:

AAGu,Ala--.Leu ---- AAGtr,Ala-.Leu "~A A Gpacking,Ala_~Leu (2) Finally, we define a 'unit volume packing energy', (A), for a given site as the ratio of the packing energy (AAGpacking between a large substituent and Ala) to the difference in their volumes (AV):

A = AAGpacking/A V

(3)

AV is calculated using the volumes occupied by the various side chains in the interiors of proteins 6. If A is characteristic of a site, it may be possible to measure its value for one substituent and use this to predict the stability for another {at the same site). For example, we can express the stability of a protein containing Leu (AGu,Leu) in terms of the stability of the protein with alanine at that site (AGu,AI~), the difference in hydrophobicities of the two side

0.025

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Values of A, the unit volume packing energy, for aliphatic substitutions in five proteins. The proteins and positions substituted are indicated across the top of the bar graph. Each group of bars shows the range of values of A calculated for that site from the available stability data. The aliphatic side chains contributing to the values of A shown by the heights of the bars are indicated: V, Vab I, lie; L, Leu.

chains (AAGtr,Ala--,Leu), and an estimate of the packing energy difference {AAGpacki.g,Ala--.Le.) between Leu and Ala at that site:

containing Ale at the site), the stability of the mutant containing the desired side chain at t h e site, and appropriate values of AGtr (Ref. 18), to calculate A A G p a c k i n g for the variAGu,Leu = AGu,Ala + AAGtr,Ala---.Leu ous aliphatic side chains at each + A A Gpacking,Ala--)Leu (4) site. We then used Eqn 3 to calculate the unit volume packing energy. The packing energy difference can If a site has a characteristic unit be estimated by rearranging Eqn 3 volume packing energy then estiand using A (estimated from a pre- mates of its value based on different vious substitution at this site) and substitutions should agree. Figure 4 the difference in volume (AV) be- shows that this is generally the case. tween Leu and Ala 6. At position 47 in gVp the unit To test the idea that interior sites volume packing energy is nearly each have a characteristic packing zero for each of three substitutions. energy, we drew upon stability Position 35 in gVp, position 75 in measurements of interior aliphatic DHFR, and position 3 in T4 lysosubstitutions in five different pro- zyme all have a negative unit volume teins (Fig. 2). For each site we evalu- packing energy for each substitution. ated A separately for each substituent These variants have stabilities lower and then compared the values of A than anticipated from the increase in (replacement of Ale129 by Val in T4 hydrophobicity when Ale is relysozyme is omitted because data placed by a larger residue. Larger from only one substitution is avail- residues do stabilize the proteins, able). We used Eqn 4, along with but these sites evidently have an AGu.Ala (the stability of the protein energetic penalty for accommodating

62

them. Finally, at positions 88 or 96 of barnase, and at position 49 of tryptophan synthase, the unit volume packing energy is always positive (i.e. proteins in which Ala is replaced by large aliphatic side chains are more stable than expected from the liquid model).

Why is the unit volume packing energy characteristic of a site? The observation that the unit volume packing energy, A, has roughly the same value for different substitutions at a given site indicates that the total packing energy is roughly proportional to the volume of the substituting residue (at least over the range encompassed by the aliphatic side chains). The packing energy reflects the difference between putting a large residue inside a protein and putting the same residue into cyclohexane. There are a number of factors that might contribute to this difference, but most of them are likely to depend on the volume of the substituting residue. The most probable cause of the energetic penalty for placing large residues at interior sites is that there simply is not enough room inside the protein. It is not surprising that this penalty should increase with increasing volume of the side chain. This has been suggested as an explanation for the smaller-than-expected stabilizations in mutants with large side chains replacing Ala at position 75 of DHFR and position 35 of gVp 23'28. Sites where Ala is the wild-type residue might be especially likely to have unfavorable packing energies upon substitution with a larger residue, if the rest of the protein is normally packed efficiently around the smaller side chain. Even if a large residue could fit inside the protein without distorting the rest of the interior, there could be an energetic penalty if the new substituent adopted an unfavorable rotational isomer in order to fit into the available space. For example, the structure of a T4 lysozyme variant in which Val replaces the wild-type Ala at position 129 has been determined 27. Valine fits inside the protein, but the side chain is in an unfavorable conformation and the mutant protein is less stable than the wild type. A different situation might occur if, for example, a protein contained a

TIBTECH- FEBRUARY 1991 [VOl.9]

cavity just the size and shape of an Ile residue, but at a site occupied by Ala. In this case, placing Ile inside the protein would be even more favorable than transferring it from water to a liquid hydrocarbon. Isoleucine would fit easily and make very good van der Waals contacts with the neighboring residues, and, unlike the liquid hydrocarbon, it would not be necessary to disrupt van der Waals contacts inside the protein to form the cavity. This has been suggested as an explanation for the high stabilities of proteins with wild-type Ile residues at positions 88 and 96 in barnase relative to mutants with Ala at these sites 24. Other properties that might affect the packing energy in a volumedependent fashion include the restriction of rotation of side chains and the packing density of protein interiors. In a protein interior, side chains are relatively fixed in position, while in a liquid hydrocarbon they could adopt many different conformations 6. This would lead to a packing energy, due to these differences in conformational entropy, that is more unfavorable for large residues, with many degrees of freedom in a liquid phase, than for Ala, which has no side chain rotational isomers. The high packing density of protein interiors leads to an opposite effect. Close packing of interior atoms means that a side chain can make favorable van der Waals contacts with more atoms than it might in a liquid hydrocarbon. Therefore large aliphatic side chains might stabilize proteins more than anticipated from liquid models 3°. In practice, these effects at least partially offset each other.

Using the unit volume packing energy for protein redesign Increasing interior hydrophobicity as a means to increase protein stability is hampered by the difficulty of determining which positions in the protein will lead to stabilization when substituted. We suggest that most interior sites in proteins have characteristic unit volume packing energies, as we have found in the cases examined here. The protein interior can then be viewed as a mosaic in three dimensions; some sites resemble an apolar liquid, others are more like a solid, and some sites are best described by a

combination of both models. This view is useful in that it can form the basis of a systematic assessment of sites in the protein most likely to lead to increased stability when substituted with large apolar residues. The first step in searching for sites of potential stabilization is to identify buried apolar residues, because increases in hydrophobicity at these positions will probably lead to the largest stabilizations. The stabilities of any two proteins containing aliphatic residues of different sizes (Ala < Val < Leu ~- Ile) are then used to calculate a rough estimate of the value of A, the unit volume packing energy, so one or two substitutions by site-directed mutagenesis will suffice for each site to be analysed. The value A can then be combined with the anticipated hydrophobicity increases to predict the effects on stability of further substitutions. In this fashion, all of the interior sites can be assessed for potential stabilization without exhaustive mutagenesis of the protein. This analysis can be used to identify sites where large aliphatic side chains will strongly destabilize, or stabilize, a proteini or alternatively, sites where the stability of the protein is independent of the nature of the amino acid side chain, and any apolar residue is tolerated.

Perspective Identifying the forces contributing to protein stability and determining their relative importance has been the focus of much research, and it was encouraging initially to find that the contribution of buried apolar residues to protein stability seemed to be a simple function of their hydrophobicities 25'26'31. The picture became less clear as the stabilities of more proteins with apolar substitutions were determined, because the dependence of stability on hydrophobicity varied from case to case 23'24'27'28. The suggestion that each interior site has a characteristic energy associated with changing the size of its occupying residue provides a conceptual framework for organizing this body of data. This view is also consistent with the picture of the protein interior provided by crystallography in which the density and composition vary with location 3.

TIBTECH -FEBRUARY 1991 [Vol. 9]

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Acknowledgements We thank D. Cheng, S. Choi and S. Wilcoxen for technical assistance; M. Horvath, H. Liang, P. Schlunk, H. Tager and H. Zabin for helpful discussions; and P. Gardner and D. Steiner for generous gifts of oligodeoxynucleotides. We a c k n o w l e d g e support (to TCT) from Merck Sharp and Dohme Laboratories, the Bristol Myers C o m p a n y , the Duchossois Foundation, the NIH and from an NSF Presidential Young Investigator Award. WSS was s u p p o r t e d by the Medical Scientist Training Program.

8 9 10

11 12 13 14

References 1 Chothia, C. (1974) Nature 248, 338-339 2 Creighton, T. E. (1984) Proteins, Freeman 3 Chothia, C. (1984) Annu. Rev. Biochem. 53,537-572 4 Chothia, C. (1975) Nature 254, 304-308 5 Richards, F. M. (1974) J. Mol. Biol. 82, 1-14 6 Richards, F. M. (1977) Annu. Rev. Biophys. Bioeng. 6, 151-176 7 Schulz, G. E. and Schirmer, R. H. []

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(1979) Principles of Protein Structure, Springer-Verlag Lesk, A. M. and Chothia, C. (1980) J. Mol. Biol. 136, 225-270 Ponder, J. W. and Richards, F. M. (1987) J. Mol. Biol. 193, 775-791 Pantoliano, M. W., Whitlow, M., Wood, J. F., Dodd, S. W., Hardman, K. D., Rollence, M. L. and Bryan, P. N. {1989) Biochemistry 28, 7205-7213 Wetzel, R. (1987) Trends Biochem. Sci. 12,478-482 Bone, R., Silen, J. L. and Agard, D. A. (1989) Nature 339, 191-195 Regan, L. and DeGrado, W. F. (1988) Science 241,976-978 DeGrado, W. F. (1988) Adv. Protein Chem. 39, 51-124 DeGrado, W. F., Wasserman, Z. R. and Lear, J. D. (1989) Science 243, 622-628 Kauzmann, W. (1959) Adv. Protein Chem. 14, 1-63 Tanford, C. (1980) The Hydrophobic Effect, Wiley Radzicka, A. and Wolfenden, R. (1988) Biochemistry 27, 1664-1670 Nozaki, Y. and Tanford, C. (1971) J. Biol. Chem. 246, 2211-2217 Pauch~re, J-L. and PliSka, V. (1983) Eur. J. Med. Chem. Chim. Ther. 18,

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Mycelial morphology and metabolite production Sergei Braun and Susan E. Vecht-Lifshitz Mycelial microorganisms are exploited extensively in the commercial production of a wide range of secondary metabolites. They can be cultured as free mycelia, as aggregated forms (pellets/flocs), or as artificially bound/entrapped cells, though problems are associated with the culture of each morphological type. Since the morphological type can strongly influence metabolite production, the methodology for inducing pellet formation, and the type of pellets produced are an important consideration for effective metabolite production. Filamentous microorganisms, moulds and actinomycetes, a c c o u n t for the majority of industrial fermentations, in terms of bulk as well as the diversity of metabolites p r o d u c e d (see Glossary) 1. These microorganisms grow as long, thin, b r a n c h e d S. Braun and S. E. Vecht-Lifshitz are at the Biotechnology Unit, Institute of Life Sciences, the Hebrew University of Jerusalem, Jerusalem 91904, Israel. © 1991, Elsevier Science Publishers Ltd (UK)

threads of m y c e l i u m . At high biomass concentration, mycelial susp e n s i o n s constitute n o n - N e w t o n i a n fluids. T h e s e are very viscous 2, except in the region near the impeller (Fig. 1). The impeller blades cause high shear stresses. In this region the broth is well aerated, but o v e r h e a t e d due to p o o r heat transfer, whereas at the p e r i p h e r y of the fermentor, the zone adjacent to the cooling surface is stagnant, oxygen-starved and over-

0167 - 9430/91/$2.00

369-375 21 Reidhaar-Olson, J. F. and Sauer, R. T. (1987) Science 241, 53-57 22 Lim, W. A. and Sauer, R. T. (1989) Nature 339, 31-36 23 Sandberg, W. S. and Terwilliger, T. C. Proc. Natl Acad. Sci. USA (in press) 24 Kellis, J. T. Jr, Nyberg, K. and Fersht, A. R. {1989) Biochemistry 28, 4914-4922 25 Yutani, K., Ogasahara, K., Tsujita, T. and Sugino, Y. (1987) Proc. Natl Acad. Sci. USA 84, 4441-4444 26 Matsumura, M., Becktel, W. J. and Matthews, B. W. (1988) Nature 334, 406-410 27 Karpusas, M., Baase, W. A., Matsumura, M. and Matthews, B. W. (1989) Proc. Natl Acad. Sci. USA ~6, 8237-8241 28 Garvey, E. P. and Matthews, C. R. (1989) Biochemistry 28, 2083-2093 29 Sandberg, W. S. and Terwillig"er, T. C. (1989) Science 245, 54-57 30 Bello, J. (1977) J. Theor. Biol. 68, 139-142 31 Kellis, J. T. Jr, Nyberg, K., Sali, D. and Fersht, A. R. (1988) Nature 333, 784-786 32 Pace, C. N. (1975) Crit. Rev. Biochem. 3, 1-43 []

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cooled (Fig. 1). Only a small part of the fermentor, v o l u m e , therefore, m a y be m a i n t a i n e d at the optimal p r o d u c t i o n conditions. Increasing the agitation rate i m p r o v e s the overall h o m o g e n e i t y , t h o u g h this also raises the p o w e r c o n s u m p t i o n and often damages the cells due to high shearing. Hence, these filamentous organisms, w i t h metabolic characteristics of c o m m e r c i a l interest, are far more difficult to use industrially than 'well-behaved' (in N e w t o n i a n terms, see Glossary) bacterial or yeast cells.

Pellets and immobilized cells Considerable i m p r o v e m e n t in the r h e o l o g y of cultures of filamentous mycelia m a y be a c h i e v e d by using either mycelial pellets (see Glossary) 2 or c o m p a c t beads of immobilized cells. On a m a c r o s c o p i c scale, a wellstirred f e r m e n t o r containing mycelial pellets seems fairly homogeneous. On a m i c r o s c o p i c scale, however, the h e t e r o g e n e i t y i m p o s e d by mass-transport limitations w i t h i n the pellet b e c o m e s apparent. Pellets 3 (see Fig. 2) and i m m o b i l i z e d cells 4

Repacking protein interiors.

Several goals of protein engineering may be achieved through redesign and repacking of protein interiors. The effects of interior apolar substitutions...
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