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Nese Sari

Publications and source records attributed to Nese Sari.

4 recordsLinked to original sources
PubMed2007

Hydrogen-deuterium exchange in free and prodomain-complexed subtilisin.

Residue-specific exchange rates of 223 amide protons in free and prodomain-complexed subtilisin were determined in order to understand how the prodomain binding affects the energetics of subtilisin folding. In free subtilisin, amide protons can be categorized according to exchange rate: 74 fast exchangers (rates > or = 1 h(-1)); 52 medium exchangers (rates between 1 h(-1) and 1 day(-1)); 31 slow exchangers (rates between 1 day(-1) and 0.001 day(-1)). The remaining 66 amide proteins did not exchange detectibly over 9 months (k(obs) < year(-1)) and were denoted as core protons. Core residues occur throughout the main structural elements of subtilisin. Prodomain binding results in high protection factors (100-1000) in the central beta-sheet, particularly in the vicinity of beta-strands S5, S6, and S7 and the connecting loops between them. These connecting loops provide the ligands to the cation at metal site B. Overall, prodomain binding seems to facilitate the organization of the entire central beta-sheet and alpha-helix C in the left-handed crossover connection between beta-strands two and three. It also appears to facilitate the isomerization of multiple prolines late in folding, allowing the formation of metal site B. The gain of stability region around site B comes at the cost of stability in regions more distal to prodomain binding: the C-terminal alpha-helix H and the N-terminal alpha-helices A and B. The acceleration of exchange in these regions by prodomain binding reveals an antagonism between the folding intermediate and the full native structure. This antagonism helps to explain why the prodomain is needed to stabilize the folding intermediate as well as why the unfolding of free subtilisin seldom occurs via this intermediate.

Amides↗
PubMed2006

Structure, dynamics, and stability variation in bacterial albumin binding modules: implications for species specificity.

Protein G-related albumin-binding (GA) modules are frequently expressed on the surfaces of bacterial cells. The limited amino acid sequence variation among GA modules results in structural and functional differences with possible implications for bacterial pathogenesis and host specificity. In particular, the streptococcal G148-GA3 and F. magna ALB8-GA albumin-binding domains exhibit a degree of structural and dynamic diversity that may account for their varied affinities for different species of albumin. To explore the impact of GA module polymorphisms on albumin binding and specificity, we recently used offset recombinant PCR to shuffle seven artificially constructed representatives of the GA sequence space and scan the phage-displayed recombinant domains for mutations that supported binding to the phylogenetically distinct human and guinea pig serum albumins (HSA and GPSA) (Rozak et al. (2006) Biochemistry 45, 3263-3271). Surprisingly, phage selection revealed an overwhelming preference for a single recombinant domain (PSD-1, phage-selected domain-1) regardless of whether the phages were enriched for their abilities to bind one or both of these albumins. We describe here the NMR-derived structure, dynamics, and stability of unbound PSD-1. Our results demonstrate that increased flexibility is not a requirement for broadened specificity, as had been suggested earlier (Johansson et al. (2002) J. Mol. Biol. 316, 1083-1099), because PSD-1 binds the phylogenetically diverse HSA and GPSA even more tightly than G148-GA3 but is less flexible. The structural basis for albumin-binding specificity is analyzed in light of these new results.

Albumins↗
PubMed2002

Solution structure of the pro-hormone convertase 1 pro-domain from Mus musculus.

The solution structure of the mouse pro-hormone convertase (PC) 1 pro-domain was determined using heteronuclear NMR spectroscopy and is the first structure to be obtained for any of the domains in the convertase family. The ensemble of NMR-derived structures shows a well-ordered core consisting of a four-stranded antiparallel beta-sheet with two alpha-helices packed against one side of this sheet. Sequence homology suggests that the other eukaryotic PC pro-domains will have the same overall fold and most of the residues forming the hydrophobic core of PC1 are highly conserved within the PC family. However, some of the core residues are predicted by homology to be replaced by polar amino acid residues in other PC pro-domains and this may help to explain their marginal stability. Interestingly, the folding topology observed here is also seen for the pro-domain of bacterial subtilisin despite little or no sequence homology. Both the prokaryotic and eukaryotic structures have hydrophobic residues clustered on the solvent-accessible surface of their beta-sheets although the individual residue types differ. In the bacterial case this region is buried at the binding interface with the catalytic domain and, in the eukaryotic PC family, these surface residues are conserved. We therefore propose that the hydrophobic patch in the PC1 pro-domain is involved in the binding interface with its cognate catalytic domain in a similar manner to that seen for the bacterial system. The PC1 pro-domain structure also reveals potential mechanisms for the acid-induced dissociation of the complex between pro- and catalytic domains.

Amino Acid Sequence↗