Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs.

Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression. Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin. Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant. In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin. The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

Eubacteria and eukaryotic cellular organelles have membrane-bound ATP-dependent proteases, which degrade misassembled membrane protein complexes and play a vital role in membrane quality control. The bacterial protease FtsH also degrades an interesting subset of cytoplasmic regulatory proteins, including sigma(32), LpxC, and lambda CII. The crystal structure of the ATPase module of FtsH has been solved, revealing an alpha/beta nucleotide binding domain connected to a four-helix bundle, similar to the AAA modules of proteins involved in DNA replication and membrane fusion. A sulfate anion in the ATP binding pocket mimics the beta-phosphate group of an adenine nucleotide. A hexamer form of FtsH has been modeled, providing insights into possible modes of nucleotide binding and intersubunit catalysis.

The effects of mutation of the conserved serine92 residue to alanine, valine, and leucine in pig myoglobin have been determined. In myoglobin crystal structures, the hydroxyl group of serine92 is within hydrogen-bonding distance of the N delta-H of histidine93, whose N epsilon coordinates the iron atom of the heme prosthetic group. The association equilibrium constants of the ferrous forms of the mutant myoglobins for O2, CO, and methyl and ethyl isocyanide are increased 1.3-13-fold relative to the wild-type protein. The rates of azide association with the mutant ferric proteins at neutral pH are decreased by factors of 2-5 consistent with an increased affinity for the iron-bound water molecule which must be displaced. The dissociation rates for azide appear to be decreased 4-10-fold, suggesting that the affinity of the mutant proteins for this ligand is also higher. Thus, the overall affinities are increased regardless of the chemical nature of the liganded species, indicating that the reactivity of the heme iron itself has been raised. Time courses for association of methyl and ethyl isocyanide at high concentrations show fast and slow phases in which the absorbance at 445 nm drops and then rises, respectively. Comparison of these traces with spectra following the reaction of isocyanide ligands with chelated proton heme in soap micelles indicates that the slow phase is associated with the breaking of the iron-proximal histidine bond and the binding of a second isocyanide species in the proximal heme pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)