Stabilization of a recombinant Fv fragment bybase-loop interconnection and VH-VL permutation

Brinkmann, Ulrich; Carlo, Angelina Di; Vasmatzis, George; Kurochkina, Natalya; Beers, Richard; Lee, Byung Kook; Pastan, Ira

doi:10.1006/jmbi.1996.0850

Cited by 31 publications

(16 citation statements)

References 32 publications

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“…Additionally, the linker sequence of (Gly) 4 Ser was also added to decrease steric hindrance of HEL during digestion by rEK. This linker sequence is flexible without a preferred structure [16,17].…”

Section: Construction Of An Expression System Of Recombinant Protein mentioning

confidence: 99%

Expression and Purification of Uniformly 15N-Labeled Amyloid β Peptide 1-40 in Escherichia coli

Nagata-Uchiyama¹,

Yaguchi²,

Hirano³

et al. 2007

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Section: Construction Of An Expression System Of Recombinant Protein mentioning

confidence: 99%

Expression and Purification of Uniformly 15N-Labeled Amyloid β Peptide 1-40 in Escherichia coli

Nagata-Uchiyama¹,

Yaguchi²,

Hirano³

et al. 2007

PPL

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“…The new termini are placed in a solvent-exposed loop, and the original ends are bridged by a peptide long enough to span the distance observed in the wild-type (WT)1 structure. These measures have produced stable and functional permutants of proteins such as GFP and related variants (4, 5), SH3 domains (6), PDZ domains (7), antibody light chain (8), and many others (e.g., refs 9-15). …”

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confidence: 99%

Structural and Thermodynamic Analysis of a Conformationally Strained Circular Permutant of Barnase

et al. 2009

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Circular permutation of a protein covalently links its original termini and creates new ends at another location. To maintain the stability of the permuted structure, the termini are typically bridged by a peptide long enough to span the original distance between them. Here, we take the opposite approach and employ a very short linker to introduce conformational strain into a protein by forcing its termini together. We join the N- and C-termini of the small ribonuclease barnase (normally 27.2 Å distant) with a single Cys residue and introduce new termini at a surface loop, to create pBn. Compared to a similar variant permuted with an 18-residue linker, permutation with a single amino acid dramatically destabilizes barnase. Surprisingly, pBn is folded at 10 °C and possesses near wild-type ribonuclease activity. The 2.25 Å X-ray crystal structure of pBn reveals how the barnase fold is able to adapt to permutation, partially defuse conformational strain, and preserve enzymatic function. We demonstrate that strain in pBn can be relieved by cleaving the linker with a chemical reagent. Catalytic activity of both uncleaved (strained) pBn and cleaved (relaxed) pBn is proportional to their thermodynamic stabilities, i.e., the fraction of folded molecules. The stability and activity of cleaved pBn are dependent on protein concentration. At concentrations above ∼2 μM, cleaving pBn is predicted to increase the fraction of folded molecules and thus enhance ribonuclease activity at 37 °C. This study suggests that introducing conformational strain by permutation, and releasing strain by cleavage, is a potential mechanism for engineering an artificial zymogen.

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“…The validity of this assertion depends on two factors: Whether the target protein is amenable to the requisite modifications (permutation and partial sequence duplication), and whether the switching mechanism can be controlled by known thermodynamic and kinetic considerations. With respect to the first point, proteins including GFP, SH3 domains, PDZ domains, antibody light chains, lysozyme, calbindin, and many others have been shown to retain structure and function after circular permutation (6,(15)(16)(17)(18)(19)(20)(21). A useful feature of AFF is that the length of the duplicated segment can be adjusted to minimize potential misfolding, degradation, or solubility problems.…”

Section: Discussionmentioning

confidence: 99%

Engineering an artificial zymogen by alternate frame protein folding

Mitrea

Parsons

Loh

2010

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

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Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two "frames" to yield the wild-type structure or a circularly permuted form in which the positions of the N-and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress k cat ∕K M by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases k cat ∕K M by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.mutually exclusive folding | design | HIV protease | molecular switch | barnase Z ymogens are inactive enzyme precursors that are activated by proteolytic cleavage. Natural zymogens play essential roles in apoptosis, blood coagulation, digestion, viral maturation, and other cellular processes. Artificial zymogens are of broad interest, with potential applications that include diagnostic tools and toxins that attack pathogens or diseased cells. Here we introduce a unique technique (alternate frame protein folding, or AFF) to convert the cytotoxic ribonuclease barnase (Bn) into an artificial zymogen that is activated by HIV-1 protease (PR). The significance of AFF is that it is general and can thus be applied to many enzymes, including those whose activities are not regulated by any existing mechanism.Several groups have previously engineered artificial zymogens. Raines and colleagues circularly permuted ribonuclease A (RNase A) and bridged the original N-and C-termini with a peptide linker that contained cleavage sequences for PR, the NS3 protease from hepatitis C, and plasmepsin II from Plasmodium falciparium (1-3). The linkers inhibited enzymatic activity by partially occluding the active site. Cleavage with the respective protease increased k cat ∕K M by approximately 100-fold. In a recent study we took a related approach by permuting Bn with a short linker peptide intended to force the termini together, thereby distorting the active site or unfolding the enzyme (4). Cleaving the linker with a chemical reagent relieved conformational strain and increased RNase activity. Although these strategies successfully created artificial zymogens, they rely on specific properties of the target enzyme and are therefore not general. Cyclic permutation typic...

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Stabilization of a recombinant Fv fragment bybase-loop interconnection and VH-VL permutation

Cited by 31 publications

References 32 publications

Expression and Purification of Uniformly 15N-Labeled Amyloid β Peptide 1-40 in Escherichia coli

Expression and Purification of Uniformly 15N-Labeled Amyloid β Peptide 1-40 in Escherichia coli

Structural and Thermodynamic Analysis of a Conformationally Strained Circular Permutant of Barnase

Engineering an artificial zymogen by alternate frame protein folding

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