Multiple Routes and Milestones in the Folding of HIV–1 Protease Monomer

Proteins fold on a time scale incompatible with a mechanism of random search in conformational space thus indicating that somehow they are guided to the native state through a funneled energetic landscape. At the same time the heterogeneous kinetics suggests the existence of several different folding routes. Here we propose a scenario for the folding mechanism of the monomer of HIV–1 protease in which multiple pathways and milestone events coexist. A variety of computational approaches supports this picture. These include very long all-atom molecular dynamics simulations in explicit solvent, an analysis of the network of clusters found in multiple high-temperature unfolding simulations and a complete characterization of free-energy surfaces carried out using a structure-based potential at atomistic resolution and a combination of metadynamics and parallel tempering. Our results confirm that the monomer in solution is stable toward unfolding and show that at least two unfolding pathways exist. In our scenario, the formation of a hydrophobic core is a milestone in the folding process which must occur along all the routes that lead this protein towards its native state. Furthermore, the ensemble of folding pathways proposed here substantiates a rational drug design strategy based on inhibiting the folding of HIV–1 protease

Preliminary X-ray studies on the GTP: AMP phosphotransferase from beef heart mitochondria

Crystals of GTP: AMP phosphotransferase from beef heart mitochondria suitable for X-ray analysis have been grown. They belong to space group I4 with unit cell dimensions: . The asymmetric unit contains two molecules each of Mr = 26,000. So far, two heavy-atom derivatives have been obtained

Mitochondrial adenylate kinase (AK2) from bovine heart Homology with the cytosolic isoenzyme in the catalytic region

The adenylate kinase isoenzyme located in the intermembrane space of mitochondria, AK2, is a monomeric protein of Mr 30000 which catalyzes the reaction ATP + AMP 〙 2ADP. 1 The protein was reduced and S‐carboxymethylated with iodo[14C2]acetate. Using a Laursen sequenator, the N‐terminal sequence of S‐carboxymethylated AK2 was determined as Ala‐Pro‐Asn‐; in some batches of the isolated protein the N‐terminal dipeptide portion was missing. The C‐terminus of AK2 was found to be Met. 2 Cleavage with CNBr yielded eight fragments which could be isolated in one step using high‐performance size‐exclusion chromatography. They ranged in size over 4–88 amino acid residues, the total being approximately 270 residues. All CNBr fragments were overlapped with Met‐containing tryptic peptides of AK2. 3 The N‐terminal 111 residues of AK2 were sequenced. Except for an N‐terminal extension of nine residues, this segment of AK2 could be aligned with the sequence 1–104 of cytosolic AK1. Allowing for two deletions in AK2, 43 of the 102 aligned residues are identical. Since this section contains the catalytic residues such as His‐36 and Asp‐93, we conclude that AK1 can serve as a three‐dimensional model of AK2 in mechanistic and drug‐designing studies. 4 Preliminary sequence results on AK2 beyond position 104 show that AK2 here contains a wing of approximately 50 residues which has no counterpart in AK1. The chain folds of the adenylate kinase isoenzymes are similar again from a position corresponding to residue 115 of AK1 onwards. The additional structural motifs of AK2 are probably related to the location of this isoenzyme in the mitochondrion

STRUCTURAL RELATIONSHIPS IN THE ADENYLATE KINASE FAMILY

The sequences of five distantly related adenylate kinases have been aligned. The local conservation of amino acids is discussed in the light of the known three-dimensional structure of one of the enzymes, the cytosolic isoenzyme 1 (AK1) from porcine muscle. The similarity profile outlines clearly the active site in the cleft of the spatial structure of AK1. The alignment reveals further that the enzyme family can be subdivided into small and large variants according to the presence or absence of a particular segment of about 30 residues in the middle of the chain. The extra segments of the large variants are strongly conserve