No abstract

The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, AG,(H,O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing AG,(H20) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed.Keywords: conformational stability; dimeric proteins; equilibrium denaturation Equilibrium denaturation studies have been very useful in understanding the structure, stabilization, and folding of small, monomeric proteins (Tanford, 1968;Pace, 1986Pace, , 1990Timasheff, 1992). The methods for analyzing the energetics of reversible unfolding of proteins by thermal or chemical denaturant techniques (Privalov, 1979;Pace, 1986;Privalov & Gill, 1988) and the conclusions that can be drawn from thorough analysis of a few proteins (Pace, 1986(Pace, , 1990Pace et al., 1990) have been presented. Many of these studies have been done with proteins whose disulfide bonds are intact, but entropic effects of the disulfide crosslinks have also been estimated (Pace, 1990). The range of stabilities calculated for monomers is between 6 and 14 kcal/mol and represents the small difference between multiple noncovalent interactions favoring the folded protein structure and unfavorable entropic terms. Such studies have been particularly useful for analysis of packing forces in protein interiors (Alber & Matthews, 1987;Matsumura et al., 1988), the testing of the globular folding of mutant proteins (Fersht et al., 1992), and the functional interaction of residues (Carter et al., Reprint requests to: Kenneth E. Neet, Department of Biological Chemistry, UHWChicago Medical School, 3333 Green Bay Road, North Chicago, Illinois 60064; e-mail: neetk@mis.fuhscms.edu. 1984). However, application of similar techniques to oligomeric proteins has, until recently, been less common, despite the potential for providing additional information on subunit interactions. Classically, the kinetics of folding pathways have been used to determine the relationship of folding to activity of oligomeric pro...

The 2.5 A X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome.

BACE1 is a key protease controlling the formation of amyloid ␤, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimer's disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academiaandindustry.Herein,wereportthenonclinicalandearlyclinicaldevelopmentofLY2886721,aBACE1activesiteinhibitorthatreached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translates in vitro activity into robust in vivo amyloid ␤ lowering in nonclinical animal models. Similar potent and persistent amyloid ␤ lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.

FAH represents the first structure of a hydrolase that acts specifically on carbon-carbon bonds. FAH also defines a new class of metalloenzymes characterized by a unique alpha/beta fold. A mechanism involving a Glu-His-water catalytic triad is suggested based on structural observations, sequence conservation and mutational analysis. The histidine imidazole group is proposed to function as a general base. The Ca(2+) is proposed to function in binding substrate, activating the nucleophile and stabilizing a carbanion leaving group. An oxyanion hole formed from sidechains is proposed to stabilize a tetrahedral alkoxide transition state. The proton transferred to the carbanion leaving group is proposed to originate from a lysine sidechain. The results also reveal the molecular basis for mutations causing the hereditary tyrosinemia type 1.