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We have determined the specificity profile of the homing endonuclease I-AniI and compared it to the conservation of its host gene. Homing endonucleases are encoded within intervening sequences such as group I introns. They initiate the transfer of such elements by cleaving cognate alleles lacking the intron, leading to their transfer via homologous recombination. Each structural homing endonuclease family has arrived at an appropriate balance of specificity and fidelity that avoids toxicity while maximizing target recognition and invasiveness. I-AniI recognizes a strongly conserved target sequence in a host gene encoding apocytochrome B and has fine-tuned its specificity to correlate with wobble versus nonwobble positions across that sequence and to the amount of degeneracy inherent in individual codons. The physiological target site in the host gene is not the optimal substrate for recognition and cleavage: at least one target variant identified during a screen is bound more tightly and cleaved more rapidly. This is a result of the periodic cycle of intron homing, which at any time can present nonoptimal combinations of endonuclease specificity and insertion site sequences in a biological host.

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Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with aminoand carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 × 10 8 sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60-80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures.Keywords: Loopentropy; random sequences; phage display Whereas the majority of multidomain proteins are formed via end-to-end linkages of domains, 28% of domains are discontinuous, suggesting that they may have evolved through the insertion of one domain into a loop of another domain (Jones et al. 1998). Specific examples include dsbA and the Escherichia coli DNA polymerase I (Russell 1994).Evolutionary benefits to forming multidomain proteins via loop insertions as opposed to end-to-end linkages would include stronger coupling between domains and increased rigidity, as the domains are linked via two connections as opposed to one, promoting allosteric interactions between the two domains.For loop insertion to be a viable evolutionary route to new proteins, it is necessary that the parent domain retains stability and function after the insertion event. Recent experiments have shown that insertions of folded domains into surface loops are generally accepted with a minimal effect on the parent domain's activity. For example, insertions of either dihydrofolate reductase (DHFR) or ␤-lactamase into four surface loops in phosphoglycerate kinase (PGK) were 197shown to have only a small...

ALD403 is a genetically engineered, humanized immunoglobulin G1 monoclonal antibody that inhibits the action of human calcitonin gene-related peptide (CGRP). Clinical trial data indicate that ALD403 is effective as a preventive therapy for migraine and has an acceptable safety profile. For preclinical characterization of ALD403, rabbit antibodies targeting a-CGRP were humanized and modified to eliminate fragment crystallizable (Fc) g receptor (FcgR) and complement interactions. The ability of ALD403 to inhibit CGRP-induced cAMP production was assessed using a cAMP bioassay (Meso Scale Discovery). The IC 50 for inhibition of cAMP release was 434 and 288 pM with the rabbit-human chimera antibody and the humanized ALD403, respectively. ALD403 inhibited a-CGRP binding with an IC 50 of 4.7 Â 10 211 and 1.2 Â 10 210 M for the a-CGRP and AMY1 receptors, respectively. ALD403 did not induce antibody-dependent cellular cytotoxicity or complementdependent cytotoxicity and did not stably interact with any of the FcgR mediating these functions, exhibiting only weak binding to FcgRI. ALD403 significantly lowered capsaicininduced blood flow responses in rodents at all time points starting at 5 minutes postapplication in a dose-dependent manner. In conclusion, ALD403 is a potent functional ligand inhibitor of a-CGRP-driven pharmacology. SIGNIFICANCE STATEMENT a-Calcitonin gene-related peptide blockade by ALD403 was assessed via radiolabeled ligand displacement, in vitro inhibition of cell signaling, and in vivo inhibition of capsaicin-induced vasodilation. Lack of engagement of fragment crystallizablemediated immune-effector functions by ALD403 was shown.