Practical applications

The neutral protease project

The role of the C-terminal amino acid in thermostability of neutral protease from Bacillus Subtilis.
V.G.H.Eijsink, B.v.d.Burg, G.Vriend, G.Venema, B.Stulp,
Prot. Engin. (1990) 3, 341.

Sub-domain interactions involved in the thermostability of neutral proteases from bacilli.
V.G.H.Eijsink, B.Stulp, B.v.d.Burg, G.Venema, G,Vriend,
Prot. Engin. (1990), 3, 341.

Contribution of the C-terminal amino-acid to the stability of Bacillus Subtilis neutral protease.
V.G.H.Eijsink, G.Vriend, B.v.d.Burg, G.Venema, B.Stulp,
Prot. Engin. (1990) 4, 99-104.

Improving the thermostability of the neutral protease of Bacillus stearothermophilus by replacing a buried asparagine by leucine.
V.G.H. Eijsink, J.R. v.d. Zee, B. v.d. Burg, G. Vriend, G. Venema,
FEBS Lt. (1991) 282, 13-16.

Thermostability of Bacillus Subtilus neutral protease.
V.G.H. Eijsink, B. v.d. Burg, G. Vriend, H.J.C. Berendsen, G. Venema,
Biochem. Int. (1991) 24, 517-525.

Stabilization of the neutral protease of Bacillus Stearothermophilus by removal of a buried water molecule.
G. Vriend, H.J.C. Berendsen, J.R. v.d. Zee, B. v.d. Burg, G. Venema, V.G.H. Eijsink,
Prot. Engin. (1991) 4, 941-945.

Introduction of a ten residue beta-hairpin in Bacillus Subtilis neutral protease.
V.G.H. Eijsink, G. Vriend, B. v.d. Burg, J.R. v.d. Zee, O.R.Veltman, B.K. Stulp, G. Venema,
Prot. Engin. (1992) 5, 157-163.

Effects of changing the interaction between domains on the thermostability of bacillus neutral proteases.
V.G.H. Eijsink, G. Vriend, B. v.d. Vinne, B. Hazes, B. v.d. Burg, G. Venema,
PROTEINS. (1992) 14, 224-236.

Increasing the thermostability of a neutral protease by replacing positively charged amino acids in the N-terminal turn of alpha-helices.
V.G.H. Eijsink, G. Vriend, B. v.d. Burg, J.R. v.d. Zee, G. Venema,
Prot. Engin. (1992) 5, 165-170.

Increasing the thermostability of the neutral protease of Bacillus Stearothermophilus by improvement of internal hydrogen bonding.
V.G.H. Eijsink, G. Vriend, J.R. v.d. Zee, B. v.d. Burg, G. Venema,
Biochem. J. (1992) 285, 625-628.

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease.
V.G.H. Eijsink, B.W. Dijkstra, G. Vriend, J.R. v.d. Zee, O.R. Veltman, B. v.d. Vinne, B. v.d. Burg, S. Kempe, G. Venema,
Prot. Engin. (1992) 5, 421-426.

Prediction and analysis of structure, stability and unfolding of thermolysin like proteases.
G. Vriend, V.G.H. Eijsink,
J. Comp. Aided Mol. Design. (1993) 7, 367-396.

Neutral protease stabilization by introduction of prolines.
F.Hardy, G.Vriend, O.R.Veltman, B.v.d.Vinne, G.Venema, V.G.H.Eijsink,
FEBS Lt. (1993) 1/2 89-92.

Structural determinants of the thermostability of thermolysin-like Bacillus neutral proteases.
V.G.H.Eijsink, G.Vriend, F.Hardy, O.R.Veltman, B.v.d.Vinne, B.v.d.Burg, B.W.Dijkstra, J.R.v.d.Zee, G.Venema,
in Stability and Stabilization of Enzymes. (1993) 91-99. W.J.J.v.d.Tweel, A.Harder, R.M.Buitelaar eds. Elsevier.

Structural determinants of the thermostability of thermolysin-like Bacillus neutral proteases.
V. Eijsink, G. Vriend, F. Hardy, O. Veltman, B. v.d. Vinne, B. v.d. Burg B. Dijkstra, J. v.d. Zee, G. Venema,
in stabilty and Stabilisation of Enzymes. 91-99. W.v.d. Tweel, A. Harder, R. Buitelaar eds. Elsevier (1993)

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease.
F. Hardy, G. Vriend, B. van der Vinne, F. Frigerio, G. Grandi, G. Venema, V.G.H. Eijsink.
Prot. Engin. (1994) 7, 425-430.

Protein stabilisation by hydrophobic interactions at the surface.
B.v.d. Burg, B. Dijkstra, G. Vriend, Bv.d. Vinne, G. Venema, V. Eijsink,
Eur. J. Biochem. (1994) 220, 981-985.

Structural determinants of the stability of thermolysin-line proteinases.
V.G.H. Eijsink, O.R. Veltman, W. Aukema,G. Vriend, G. Venema.
Nature Struct. Biol. (1995) 2, 374-379

Introduction of disulfide bonds into bacillus stearothermophilus neutral protease and effects on its thermal stability.
J.Mansfeld, G. Vriend, B. Dijkstra, G. Venema, R.Ulbrich-Hofmann, V.G.H. Eijsink,
in Perspectives on Protein Engineering, M.J. Geisow, R. Epton Eds. Mayflower Worldwide Ltd. Birmingham, (1995) pp 205-206.

Analysis of structural determinants of the stability of thermolysin like proteases by molecular modelling and site-directed mutagenesis.
O.R. Veltman, G. Vriend, H. Middelhoven, B. van den Burg, G. Venema, V. Eijsink
Prot.Eng. (1996) 9, 1181-1189.

Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond.
J.Mansfeld, G.Vriend, B.W.Dijkstra, O.R.Veltman, B.v.d.Burg, G.Venema, R.Ulbrich-Hofmann, V.G.H.Eijsink,
JBC (1997) 272, 11152-11156.

There are many ways to skin a cat, and many ways to make a protein more stable. One of the main goals of the neutral protease project has always been to make them more stable. We knew that local unfolding processes followed by autolysis of these locally unfolded loops are the reason for the denaturation of neutral proteases at elevated temperatures. Therefore we have put a lot of effort into finding out which is the loop that unfolds first because that loop is obviously the weakest link and stabilising that loop is the only thing that really makes sense. From the other articles on this page you can see that through the years we homed in on the area around calcium 3 in the N-terminal domain of TLP-ste as being this weakest link. The introduction of a cys-cys bridge that connects this weakest link with another part of the molecule seemed to us the most straightforward way of avoiding this local unfolding. Indeed, this article shows that we were right because a cys-cys bridge 'at the right spot' made TLP-ste a lot more stable.

Engineering thermolysin-like proteases whose stability is largely independent of calcium.
O.R.Veltman, G.Vriend, B.v.d.Burg, F.Hardy, G.Venema, V.G.H.Eijsink,
Febs Lt. (1997) 405, 241-244.

The stability of neutral proteases depends on the calcium concentration. A low calcium concentration leads to local unfolding at a lower temperature of a loop (or more loops) near calcium ions (or actually, near the positions where the calciums once were, because we have good reasons te believe that the calcium containing loops unfold only after the calcium goes on vacation). So, if we make a mutation that pesters the calcium out, we should get the same stability as at very low calcium concentrations. In this article we show this to be the case, and we show how our understanding of the atomic interactions around calcium 3 allowed us to compensate for the stability loss by making a few more mutations in the same region of the molevule.

Model building of a thermolysin-like protease by mutagenesis.
F.Frigerio, I.Margarit, R.Nogarotto, G.Grandi, G.Vriend, F.Hardy, O.R.Veltman, G.Venema, V.G.H.Eijsink
Prot. Engin. (1997) 10, 223-230.

This article is described in the modelling methos section.

Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases.
O.R.Veltman, G.Vriend, F.Hardy, J.Mansfeld, B.v.d.Burg, G.Venema, V.G.H.Eijsink,
EJB. (1997) 248, 433-440.

We know that local unfolding processes followed by autolysis of these locally unfolded loops are the reason for the denaturation of neutral proteases at elevated temperatures. Therefore we have put a lot of effort into finding out which is the loop that unfolds first because that loop is obviously the weakest link and stabilising that loop is the only thing that really makes sense. From the other articles on this page you can see that through the years we homed in on the area around calcium 3 in the N-terminal domain of TLP-ste as being this weakest link. In this article we describe more than twenty mutations in the area around calcium 3. Not only does this study confirm the importance of this area for thermal stability, the detailed analysis also forms the basis for the production of proteases that are extremely stable under a wide variety of experimental conditions.

Engineering an enzyme to resist boiling.
B.v.d.Burg, G.Vriend, O.R.Veltman, G.Venema, V.G.H.Eijsink,
PNAS. (1998) 95, 2056-2060.

This is one of the tops in the neutral protease roler coaster ride. Here we actually combined most of the knowledge we gathered in the articles listed above and managed to make one of the neutral proteases boilable. See also the commentary in the same volume of PNAS:
Frances H. Arnold Enzyme engineering reaches the boiling point PNAS 1998 95: 2035-2036. The text of this press-release looks like:
Researchers Create Artificial Enzyme to Resist Harsh Conditions
By making only a handful of structural changes to a naturally occurring enzyme, scientists now have created a "hyperstable" artificial enzyme that retains its enzymatic activity at boiling temperatures and in the presence of normally destructive chemicals. Using computer modeling techniques to predict the effects of a huge number of enzyme changes, the multidisciplinary team identified eight mutations that had the greatest impact on the stability or "staying power" of the enzyme under extreme heat conditions and exposure to harsh chemicals. In the article beginning on Page 2,056, they report that the engineered enzyme incorporating these mutations is more than 340 times as stable at the boiling point as its natural counterpart. Furthermore, the mutant protein remains active even at high concentrations of denaturing or destructive agents. Most remarkable, the authors note, is that the performance of the artificial enzyme is not compromised at lower temperatures. While hyperstable enzymes exist in nature, they typically have significantly reduced activity at low temperatures. These results have clear applications in farming, in which enzymes help to predigest animal feed and make use of certain leftovers, and in leather processing, where they can convert environmentally toxic residuals into safer byproducts. The researchers are from the University of Groningen, European Molecular Biology Laboratory, BIOcomputing and Agricultural University of Norway. For further information, contact Bertus Van den Burg at the University of Groningen in the Netherlands; tel. 31 50 3632102, fax 31 50 3632348, or e-mail .

Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission.
B.van den Burg, V.G.H. Eijsink, G. Vriend, O.R. Veltman, G. Venema Biotechnol.
Appl. Biochem. 27, in press (1998)

Sometimes you try to avoid being bitten by the dog and than you get scratched by the cat. Under somewhat 'funny' conditions a few of the cleavage sites in NP-sub could be detected. We did not know if these were the same sites as the ones that are in our way on the path to greater stability for NP-sub, but we tried anyway to eliminate one site by mutagenesis. This site was at the extremity of the active site pocket, and, since mobility of the active site seems important (see above) for proper functioning of the enzyme, the mutant design required some thinking. Well, we succeeded. The cleavage site is gone. However, in the mutant a new cleavage site is observed, just a turn of a helix away and the achieved stabilisation is marginally. You cannot win them all. The statement "Once you know why the protein is unstable, the stability problem is solved" got confirmed one more time.

Manipulating the autolytic pathway of a Bacillus protease.
B. Van den Burg, V.G.H. Eijsink, G. Vriend, O.R. Veltman, G. Venema
In Proteolysis in Cell Functions (V.K. Hopsu-Havu et al., eds.), IOS Press, 1997.

This is a chapter in a book giving an overview of what we have done to alter the autolysis (and thus the stability) of neutral proteases.

Manipulating Bacillus neutral protease autolysis.
B. Van den Burg, V.G.H. Eijsink, G. Vriend, O.R. Veltman, G. Venema 1997,
Prot.Eng. 10, 37.

This is a published poster abstract for a meeting on protein engineering.

Identification of the critical calcium site in the thermolysin-like protease of B.Stearothermophilus reveals initial steps in the thermal unfolding.
O.R. Veltman, V.G.H. Eijsink, G. Vriend, G. Venema, B. Van den Burg 1997,
Prot.Eng. 10, 37.

This is a published poster abstract for a meeting on protein engineering.

The uteroglobin project

Identification of residues essential for progesteron binding to uteroglobin by site-directed mutagenesis.
W. Peter, H-J. Brueller, G. Vriend, M. Beato, G. Suske,
J. Steroid Biochem. Molec. Biol. (1991) 38, 27-33.

Interchain cysteine bridges control entry of progesteron to the central cavity of the uteroglobin dimer.
W. Peter, R. Dunkel, P.W.F. Stouten, G. Vriend, M. Beato, G. Suske,
Prot. Engin. (1992) 5, 351-359.

Progesterone can bind Uteroglobin in two conformations.
R. Dunkel, G. Vriend, G. Suske.
Prot. Engin. (1995) 8, 71 -79.

The carboxypeptidase project

Model building of the enzymatically active subunit of human serum carboxypeptidase-N.
D. Hendriks, M. Vingron, G. Vriend, W. Wang, S. Scharpe.
in Archives Internationales de Physologie, de Biochemie et de Biophysique. (1992) 100, 13.

Comparative molecular modelling of the active site human kininase-I.
D. Hendriks, M. Vingron, G. Vriend, W. Wang, D. Nalis, S. Scharpe.
in Agents and Actions, supplement. Krikhauser, Batel. (1992) Vol. 1.

On the specificity of Carboxypeptidase N, a Comparative Study.
D.Hendriks, M.Vingron, G.Vriend, W.Wang, D.Nalis, S.Scharpe,
Biol. Chem. Hoppe-Seyler (1993) 374, 943-849.

Comparative model building of plasma carboxypeptidase U.
D. Hendriks, G. Vriend, W. Wang, S. Scharpe,
in Fibrinolysis, C. Livingstone, J. Davidson, I. Walker, eds. (1995) pp. 39.

Other projects

Structure function relation for Crotoxin from Crotalus Durissus Terificus.
Y.P. Mascarenhas, C.J. Laure, P. Stouten, G.Vriend. Eur.
Biophys. J. (1992) 21, 199-205.

Refinement of TIM at 1.8 A resolution.
R. Wierenga, M. Noble, J. Zeelen, G. Vriend, S. Nauche, W.G.J. Hol,
J. Mol. Biol. (1991) 220, 995-1015.

Proteases and their inhibitors: Today and tomorrow.
S.Scharpe, I. De Meester, D. Hendriks, G. Vanhoof, M. v. Sande, G. Vriend,
Biochimie, (1991) 73, 121-126.

A transactivation pathway that requires a direct interaction between an embryonal carcinoma cell-specific factor and the TATA-binding protein.
M.Keaveney, A.Berkenstam, G.Vriend, H.G.Stunnenberg.
Nature (1993) 365, 562-566.

Molecular characterisation of an extracellular acid resistant lipase produced by Rhizopus javanicus.
W. Uyttenbroeck, D. Hendriks, G. Vriend, I. Debaere, L. Moens, S. Scharpe.
Biol. Chem. Hoppe Zeiler (1993) 374, 245 - 254.

Cloning and sequence analysis of human lymphocyte prolyl endopeptidase.
G. Vanhoof, F. Goossens, L. Hendriks, I. de Meester, D. Hendriks, G. Vriend, C. van Broeckhoven, S, Scharpe.
Gene (1994) 149, 363-366.

The role of electrostatic charge in the membrane insertion of Colicin A: Calculation and mutation.
J.H. Lakey, M.W. Parker, J.M. Gonzalez-Manas, D. Duche, G. Vriend, D. Baty, F. Pattus,
Eur. J. Biochem. (1994) 220, 155-163.

The N-terminal domain of VirG of Agrobacterium tumefaciens: modelling and analysis of mutant phenotypes.
K. Rodenburg, E. Scheeren-Groot, G. Vriend, P. Hookaas
Prot. Engin. (1994) 7, 905-909.

Electrostatics and the unfolding of colicins at the membrane surface.
J.H.Lakey, M.A.Parker, J.-M. Gonzales-Manas, D.Duche, G.Vriend, D.Baty, F.Pattus, (1994)
in: Bacterial Toxins, Zbl. Bakt. Suppl. 24, 361-362. eds Freer et al. Gustav Fisher, Stuttgart, Jena, New York.

The purification, characterisation and analysis of primary and secondary structure of prolyl oligopeptidase from human lymphocytes.
F. Goossens, I. De Meester, G. Vanhoof, D. Hendriks, G. Vriend, S. Scharpe
Eur. J. Biochem. (1995) 233, 432-441.

ACGT and vicilin ciore sequences in a promotor domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein.
Vincentz,M., Leite,A., Neshich,G., Vriend,G,, Mattar,C,, Barros,L., Weinberg,D., Almeida,E.R.de, Carvalho,M.P.de, Aragao,F., Gander,E.S.,
Plant Mol.Biol. (1997) 34, 879-889.

This is an example of protein DNA interaction modelling.

Analysis of the black-eyed pea trypsin and chymotrypsin inhibitor-alpha-chymotrypsin complex.
Freitas,S.M.de, Mello,L.V.de, Silva,M,C,da, Vriend,G., Neshich,G., Ventura,M.M.,
FEBS Lett. (1997) 409, 121-127.

The big thing about this article is that the two template BBI structures (one solved by NMR, onme by Xray) are both 'wrong' by all thinkable structure validation standards. The Brazilian partners in this project figured out that the protein is multimeric. Something the NMR spectroscopists totally missed....

The Mutations in FGFR2 Associated Craniosynostoses are Clustered in Five Structural Elements of Immunoglobulin-like Domain III of the Receptor"
D.Steinberger, G.Vriend, J.Mulliken, U.Mueller,
Human Genetics (1998) 102: 145-150

New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2.
V.Botquin, H.Hess, G.Fuhrmann, C.Anastasiadis, M.K.Gross, G.Vriend, H.R.Schoeler
Genes and Developement. (1998) probably accepted.

Experimental evidence suggested that the OCT4 - DNA complex could very well contain two OCT4 molecules. A modelling study indicated that this was a very plausible suggestion, and the model suggested some mutations that could be made to validate the hypothesis. The fact that this article got submitted strongly indicates that the mutations were succesful...

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutant can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power.
J.C. Williams, J.P.Zeelen, G.Neubauer, G.Vriend, JBackmann, P.A.M.Michels, A-M.Lambeir, R.K.Wierenga,
Prot.Eng. (1999) 12, 243-250.

A Glu on the dimer interface of TIM is not very happy. It has to be protonated to sit there at all. This protonation costs a lot of energy (we are working on a computational estimate of how much it costs). Mutating it to Gln (which is at that position in many other TIMs, makes the thing darn stable.