Combining computational and experimental screening for rapid optimization of protein properties

Hayes, Robert J.; Bentzien, Jörg; Ary, Marylouise; Hwang, Marian Y.; Jacinto, Jonathan; Vielmetter, Jost; Kundu, Anirban; Dahiyat, Bassil I.

doi:10.1073/pnas.212627499

Cited by 105 publications

(92 citation statements)

References 50 publications

(50 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The application of random mutagenesis did not yield either one of the rational design variants rTK2 or rTK3. The result can be explained by the fact that an Arg104Gln change is possible by a single nucleotide substitution (CGA to CAA) while Arg104Met (CGA to ATG) requires simultaneous nucleotide changes in all three codon positions (29). Confronted with the possibility that alternative amino acid substitutions in these two residues could produce enzymes with tk activity, we applied site-saturation mutagenesis to completely randomize both positions.…”

Section: In Vitro Characterization Of Selected Dck Mutants From Randomentioning

confidence: 99%

Systematic Exploration of Active Site Mutations on Human Deoxycytidine Kinase Substrate Specificity

Iyidogan¹,

Lutz²

2008

Biochemistry

View full text Add to dashboard Cite

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analogue prodrugs in addition to its natural substrates, 2′-deoxycytidine, 2′-deoxyguanosine, and 2′-deoxyadenosine. To improve the low catalytic activity and tailor the substrate specificity of dCK, we have constructed libraries of mutant enzymes and tested them for thymidine kinase (tk) activity. Random mutagenesis was employed to probe for amino acid positions with an impact on substrate specificity throughout the entire enzyme structure, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. Kinetic analysis indicates that Arg104Gln/Asp133Gly creates a "generalist" kinase with broader specificity and elevated turnover for natural and prodrug substrates. In contrast, the substitutions of Arg104Met/ Asp133Thr, obtained via site-saturation mutagenesis, yielded a mutant with reversed substrate specificity, elevating the specific constant for thymidine phosphorylation by over 1000-fold while eliminating activity for dC, dA, and dG under physiological conditions. The results illuminate the key contributions of these two amino acid positions to enzyme function by demonstrating their ability to moderate substrate specificity.Human deoxycytidine kinase (dCK; 1 EC 2.7.1.74) catalyzes the phosphorylation of 2′-deoxycytidine (dC), 2′-deoxyadenosine (dA) and 2′-deoxyguanosine (dG) to their corresponding monophosphates using nucleoside triphosphates as phosphoryl donors. This reaction is the first step of the deoxyribonucleoside salvage pathway, an alternative to de novo nucleotide biosynthesis, which, in combination with deoxyribonucleoside mono-and diphosphate kinases, provides triphosphate anabolites for DNA replication and repair (1).In addition to recycling natural 2′-deoxyribonucleosides, dCK catalyzes the initial, often ratedetermining phosphorylation of several chemotherapeutic nucleoside analogue (NA) prodrugs such as gemcitabine (2′,2′-difluorodeoxycytidine), AraC (1-β-D-arabinosylcytosine), and clofarabine [2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-9H-purin-6-amine], as well as antiviral prodrugs including ddC (2′,3′-dideoxycytidine), 3TC (2′-deoxy-3′-thiacytidine), and FTC (5-fluoro-2′-deoxy-3′-thiacytidine) whose pharmacological activity depends on their triphosphate form (2-7). Given the critical role of dCK in phosphorylating 2′- † The authors would like to acknowledge financial support in part by NIH Grant GM69958 and by a grant to the Emory Center for AIDS Research (AI050409) from the NIH and by institutional funding from the Emory University Health Science Center. *Corresponding author: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322. Tel: (404) 712-2170. Fax: (404) 727-6586. E-mail: sal2@emory.edu. SUPPORTING INFORMATION AVAILABLE Oligonucleotide sequences used as primers for the site-directed mutagenesis (Table S-1) and statistical data on the codon distribution of the site-saturation mutage...

show abstract

Section: In Vitro Characterization Of Selected Dck Mutants From Randomentioning

confidence: 99%

Systematic Exploration of Active Site Mutations on Human Deoxycytidine Kinase Substrate Specificity

Iyidogan¹,

Lutz²

2008

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Although many algorithms have now been proposed to design such combinatorial libraries (7-9, 11, 12), few computationally designed libraries have been characterized experimentally (9,18,19), and, to our knowledge, there have been no controlled experiments comparing these methods with each other or with libraries of randomly generated sequence diversity. The results of such a comparison would be hard to predict, especially because none of these methods models protein function explicitly.…”

mentioning

confidence: 99%

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Treynor

Vizcarra²,

Nedelcu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.GFP ͉ library design ͉ protein design ͉ protein engineering ͉ high-throughput screening P rotein sequence space is so vast that one can easily imagine the optimal sequence for a particular application will never be sampled by random mutation and recombination. Structure-based computational protein design tools seek to screen that sequence space more thoroughly than can be screened in the laboratory, but are currently based on approximate representations of candidate sequences and an incomplete understanding of the relationships between structure and function. Although many algorithms used to screen sequences in silico aim to identify a single optimal sequence (1-5), others aim instead to optimize the composition of a library of sequences (6-13). Provided that resources exist to synthesize and screen such libraries, library design algorithms compensate for the approximations built into them by increasing the number of attempts at designing the desired function. Viewed from a complementary perspective, such algorithms aim to sample sequence space more effectively than methods that randomly generate sequence diversity.Designed libraries can be synthesized for roughly the same cost as a designed sequence by recognizing the opportunities in gene synthesis for the combinatorial shuffling of sequence diversity (14-17). Although many algorithms have now been proposed to design such combinatorial libraries (7-9, 11, 12), few computationally designed libraries have been characterized experimentally (9,18,19), and, to our knowledge, there have been no controlled experiments comparing these methods with each other or with libraries of randomly generated sequence diversity. The results of such a comparison would be hard to predict, especially because none of these methods models protein function explicitly. Instead, these algorithms attempt to model protein stability as a surrogate for protein function on the assumption that libraries with a greater fraction of well folded proteins are more likely to contain variants with the desired function.Here, we evaluate seven design...

show abstract

“…The success of the semidirected library, which was based only on considerations of E6AP stability, suggests that enriching with well-folded sequences was the critical step for identifying binders. Computationally enriching for folded protein has similarly been shown to aid the selection of proteins with previously undescribed fluorescent properties and improved enzymatic activity (23,30,31).…”

Section: Discussionmentioning

confidence: 99%

Engineering a protein–protein interface using a computationally designed library

Guntas

Purbeck

Kuhlman

2010

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Computational algorithms for protein design can sample large regions of sequence space, but suffer from undersampling of conformational space and energy function inaccuracies. Experimental screening of combinatorial protein libraries avoids the need for accurate energy functions, but has limited access to vast amounts of sequence space. Here, we test if these two traditionally alternative, but potentially complementary approaches can be combined to design a variant of the ubiquitin-ligase E6AP that will bind to a nonnatural partner, the NEDD8-conjugating enzyme Ubc12. Three E6AP libraries were constructed: (i) a naive library in which all 20 amino acids were allowed at every position on the target-binding surface of E6AP (13 positions), (ii) a semidirected library that varied the same residue positions as in the naive library but disallowed mutations computationally predicted to destabilize E6AP, and (iii) a directed library that used docking and sequence optimization simulations to identify mutations predicted to be favorable for binding Ubc12. Both of the directed libraries showed >30-fold enrichment over the naive library after the first round of screening with a split-dihydrofolate reductase complementation assay and produced multiple tight binders (K d < 100 nM) after four rounds of selection. Four rounds of selection with the naive library failed to produce any binders with K d 's lower than 50 μM. These results indicate that protein design simulations can be used to create directed libraries that are enriched in tight binders and that in some cases it is sufficient to computationally screen for well-folded sequences without explicit binding calculations.computational protein design | protein engineering | Rosetta C omputational design and selection from combinatorial libraries are alternative approaches for protein engineering. In computational design, high-resolution models of protein structure are used to predict amino acid sequences that will stabilize target protein structures or complexes. Computational design has been used to stabilize protein structures and interfaces, redesign protein-binding specificities, and in a few cases build protein structures from scratch (1-4). Despite these successes, many problems in protein engineering remain very difficult for computational design: These include the design of protein-protein interactions and the creation of enzymes that compare favorably with naturally occurring enzymes (5-9). In silico ranking of designed interfaces is challenging because large free energies of desolvation have to be correctly balanced by hydrogen bonding and van der Waals interactions that are sensitive to small changes in atomic positions. As a result, there are often significant errors in calculated energies for protein-binding events. One advantage of screening or selecting from large-scale combinatorial libraries is that it does not rely on the accuracy of an energy functioneach sequence is experimentally tested for the desired functionality. Protein selection techniques such as...

show abstract

Combining computational and experimental screening for rapid optimization of protein properties

Cited by 105 publications

References 50 publications

Systematic Exploration of Active Site Mutations on Human Deoxycytidine Kinase Substrate Specificity

Systematic Exploration of Active Site Mutations on Human Deoxycytidine Kinase Substrate Specificity

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Engineering a protein–protein interface using a computationally designed library

Contact Info

Product

Resources

About