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Journal Issue - Volume 10 Issue 1 (January 2001)

Abstract The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein...

Abstract The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (PfuPCNA), at 2.1 Å resolution. PfuPCNA forms a toroidal, ring‐shaped structure consisting of homotrimeric molecules, which...

Abstract The leucine zipper is a dimeric coiled‐coil structural motif consisting of four to six heptad repeats, designated (abcdefg)n. In the GCN4 leucine zipper, a position 16 in the third heptad is occupied by an Asn residue whereas the other a positions are Val residues. Recently, we have constructed variants of the GCN4 leucine zipper in which the a position Val residues were replaced by Ile. The folding and unfolding of the wild‐type GCN4 leucine zipper and the...

Abstract The binding mechanism of Mg2+ at the M3 site of human placental alkaline phosphatase was found to be a slow‐binding process with a low binding affinity (KMg(app.) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg2+‐free and Mg2+‐containing enzymes by acrylamide showed almost identical dynamic quenching constant (Ksv = 4.44 ± 0.09 M−1), indicating that there is no gross conformational difference between the M3‐free and the M3‐Mg2+...

Abstract Proteins in the molten globule state contain high levels of secondary structure, as well as a rudimentary, nativelike tertiary topology. Thus, the structural similarity between the molten globule and native proteins may have a significant bearing in understanding the protein‐folding problem. To explore the nature of side‐chain–side‐chain interactions in the α‐lactalbumin (α‐LA) molten globule, we determined the effective...

Abstract The structure of Herpes simplex virus type 1 thymidine kinase (TKHSV1) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TKHSV1, measured by thermal denaturation...

Abstract Calerythrin, a four‐EF‐hand calcium‐binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca2+ and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near‐UV CD bands decreases. Yet far‐UV CD spectra indicate only a small decrease in the amount of secondary...

Abstract The fold of the murine Sox‐5 (mSox‐5) HMG box in free solution has been determined by multidimensional NMR using 15N‐labeled protein and has been found to adopt the characteristic twisted L‐shape made up of two wings: the major wing comprising helix 1 (F10–F25) and helix 2 (N32–A43), the minor wing comprising helix 3 (P51–Y67) in weak antiparallel association with the N‐terminal extended segment. 15N relaxation measurements show...

Abstract DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the...

Abstract Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three‐dimensional structure of...

Abstract As a step toward selecting folded proteins from libraries of randomized sequences, we have designed a ‘loop entropy reduction’‐based phage‐display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable...

Abstract We have carried out molecular dynamics simulations of the native dihydrofolate reductase from Escherichia coli and several of its folded protein fragments at standard temperature. The simulations have shown fragments 1–36, 37–88, and 89–159 to be unstable, with a CαRMSD (Cα root mean squared deviation) >5 Å after 3.0 nsec of simulation. The unfolding of fragment 1–36 was immediate, whereas fragments 37–88 and 89–159 gradually...

Abstract Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross‐links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross‐links, protein flexibility in the vicinity of a cross‐link, and disulfide loop size on the stability of Cucurbita maxima trypsin...

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