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Long Loop (long + loop)

Distribution by Scientific Domains

Chemistry	100%

Selected Abstracts

The kinetics of G-CSF folding

PROTEIN SCIENCE, Issue 10 2002
David N. Brems
Abstract The folding kinetics of G-CSF were determined by trp-fluorescence and far-UV circular dichroism. Folding and unfolding was achieved by rapid dilution and mixing of the denaturant, GdnHCl. G-CSF is a four-helical bundle protein with two long loops between the first and second helices and between the third and fourth helices. The entire conformational change expected by fluorescence was observed by stopped-flow technology, but due to rapid refolding kinetics only a portion was observed by circular dichroism. G-CSF contains two trp residues, and their contribution to the fluorescent-detected kinetics were deciphered through the use of single-site trp mutants. The trp moieties are probes of the local conformation surrounding their environment. One trp at residue 118 is located within the third helix while the other trp at residue 58 is part of the long loop between the first and second helices. The refolding results were most consistent with the following mechanism: U , I1 , I2 , N; where U represents the unfolded protein, I1 represents intermediate state 1, I2 represents intermediate state 2, and N represents the native state. I1 is characterized as having approximately one-half of the native-like helical structure and none of the native-like fluorescence. I2 has 100% of the native helical structure and most of the trp-118 and little of the trp-58 native-like fluorescence. Thus refolding occurs in distinct stages with half of the helix forming first followed by the remaining half of the helix including the third helix and finally the loop between the first and second helices folds. [source]

Structures of hypoxanthine-guanine phosphoribosyltransferase (TTHA0220) from Thermus thermophilus HB8

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010
Mayumi Kanagawa
Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase), which is a key enzyme in the purine-salvage pathway, catalyzes the synthesis of IMP or GMP from ,- d -phosphoribosyl-1-pyrophosphate and hypoxanthine or guanine, respectively. Structures of HGPRTase from Thermus thermophilus HB8 in the unliganded form, in complex with IMP and in complex with GMP have been determined at 2.1, 1.9 and 2.2,Å resolution, respectively. The overall fold of the IMP complex was similar to that of the unliganded form, but the main-chain and side-chain atoms of the active site moved to accommodate IMP. The overall folds of the IMP and GMP complexes were almost identical to each other. Structural comparison of the T. thermophilus HB8 enzyme with 6-oxopurine PRTases for which structures have been determined showed that these enzymes can be tentatively divided into groups I and II and that the T. thermophilus HB8 enzyme belongs to group I. The group II enzymes are characterized by an N-terminal extension with additional secondary elements and a long loop connecting the second ,-helix and ,-strand compared with the group I enzymes. [source]

Low free energy cost of very long loop insertions in proteins

PROTEIN SCIENCE, Issue 2 2003
Michelle Scalley-Kim
Abstract 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 amino- and 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 × 108 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. [source]