Extensive Secondary Structure Research Articles

Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: complete 1H, 15N and 13C backbone assignments and secondary solution structure of the flavodoxin.

The 1H, 15N and 13C backbone and 1H and 13C beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30 degrees C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, 3JHNH alpha coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel beta-sheet and five alpha-helices. One of the outer regions of the beta-sheet shows no regular extended conformation, whereas the outer strand beta 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five alpha-helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the alpha-helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn11, Ser68 and Asn72 are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.

Site-directed mutagenesis using overlap extension PCR.

Site-directed mutagenesis and the polymerase chain reaction (PCR) represent two powerful techniques that have led to rapid advances in our understanding of gene expression and function. Early protocols for site-directed mutagenesis depended on the production of single-stranded DNA containing the gene of interest (1), using Ml 3 phage, or phagemids such as pBluescript. One limitation with this method is the presence of inverted repeat sequences and other complementary regions, which form extensive secondary structures within single-stranded DNA, which often severely decrease the ability to extend annealed primers containing the mutation of interest. Recently, several groups have described PCR-based site-directed mutagenesis techniques that avoid this problem by using thermal stable DNA polymerases and high temperatures to melt secondary structure (2— 4). Although several variations of these techniques have been reported, this chapter focuses on a derivative of the Sarkar and Sommer (4) megaprimer method termed Overlap Extension Mutagenesis, which is schematically depicted in Fig. 1. In the first round of PCR, primer A and mutagenic primer B are used to amplify the region of interest as well as introduce the desired mutation. This amplified product is then used as a primer in the second round of PCR and, as such, is referred to as a megaprimer. In the second round of PCR, megaprimer A/B and primer C

Multiple Species of Defective RNAs in Plants Infected with Citrus Tristeza Virus

Alemow (Citrus macrophylla) and sweet orange (C. sinensis) plants infected, respectively, with several Israeli and Florida isolates of the citrus tristeza virus (CTV) were found to contain multiple species of RNA molecules with features similar to defective-interfering RNAs. Northern blot hybridizations of dsRNAs extracted from serial passages of the Israeli VT isolate (CTV-VT) and from different plants infected with a single source of inoculum showed considerable variation both in the presence and in the relative abundance of the defective RNA (D-RNA) bands. The D-RNA molecules were found to be encapsidated in the CTV particles. Sequence analysis of two VT D-RNA molecules of 2.7 and 4.5 kb revealed that they were composed of two regions corresponding to 1818 and 4036 nucleotides from the 5′ and 938 and 442 nucleotides from the 3′ termini of the CTV-VT genomic RNA, respectively. A short (ca. 0.8 kb) nonencapsidated single-stranded positive-sense RNA species was also found in infected plants. This ssRNA, which copurified with dsRNAs, was shown by hybridization to encompass the 5′-terminal part of the CTV genome and might have an extensive secondary structure.

Regulation of the Caulobacter crescentus dnaKJ operon.

The bacterial heat shock proteins DnaK and DnaJ are members of a class of molecular chaperones that are required for a wide variety of cellular functions at normal growth temperatures. In Caulobacter crescentus, the expression of the dnaKJ operon is regulated both temporally during the normal cell cycle and by heat shock. Analysis of deletions and base substitutions in the 5' region of the operon established the presence of two functional promoters: a heat shock-inducible promoter, P1, with characteristics of a sigma 32 promoter, and an adjacent sigma 70-like promoter, P2. Transcription initiating at the sigma 70-like promoter is under strict temporal control, whereas transcription initiating at the heat shock promoter at 30 degrees C is not. Transcription of dnaKJ occurs during a short period in the cell cycle, concomitant with the onset of DNA replication. Deletions in the 5' region have also revealed that all cis-acting sites required for temporal control of transcription reside within 50 bases of the P2 start site. Transcripts initiating from either the P1 or the P2 promoter have an RNA leader sequence with a high probability of forming an extensive secondary structure. Deletion of this leader sequence resulted in an increased rate of expression in both transcriptional and translational fusions. Although the temporal control of expression at physiological temperatures is not affected by the presence or absence of the leader sequence, changes in mRNA secondary structure may contribute to the modulation of DnaK and DnaJ levels at normal temperatures and during heat shock.

Sequence of RNA2 of the Helicoverpa armigera stunt virus (Tetraviridae) and bacterial expression of its genes.

The complete nucleotide sequence of RNA2 of Helicoverpa armigera stunt virus (HaSV), a member of the Tetraviridae, was determined by characterization of cloned cDNA and PCR products and direct sequencing of genomic RNA. The capped, positive sense, single-stranded RNA is 2478 nucleotides in length and has two overlapping open reading frames (ORFs) likely to be cistrons which are situated between terminal non-coding regions of 282 and 168 bases, 5' and 3', respectively. Extensive secondary structure of the RNA strand is indicated, including a tRNA-like structure at the 3' terminus which is the first such structure discerned in an animal virus. The first ORF encodes a 17 kDa PEST protein (p17) of unknown function while the second ORF encodes the 71 kDa coat protein precursor (p71) that is cleaved at an Asn-Phe site into the 64 kDa and 7 kDa coat proteins. The precursor coat protein is 66% identical to that of another tetravirus, the Nudaurelia omega virus, with most of the difference residing in a 165 amino acid region located in the middle of the sequence. Despite the extensive similarity, no serological relationship was observed between the two viruses, suggesting that the dissimilar region is exposed on the capsid exterior. Expression in bacteria of the two RNA2 gene products shows they are likely to be expressed by a leaky scan-through mechanism. Bacterial expression of p71 did not produce virus-like particles while expression of p17 produced large arrays of mostly hollow, hexagonal tube-like structures.

Thermodynamic and functional characterization of a stable IgG conformer obtained by renaturation from a partially structured low pH-induced state

At pH 2, rabbit IgG adopts a partially structured state that exhibits loss of thermal unfolding transition, tentatively assigned to the CH2 domain, whilst retaining a well-defined tertiary structure for the rest of the molecule and extensive secondary structure. Renaturation of IgG from this state yields a stable conformer that differs from native IgG by a lower degree of interaction between the CH2 and CH3 domains, and stronger interaction between the CH1 and CH2 domains, as judged by differential scanning calorimetry and probing the IgG conformation with specific ligands (C1q component of complement, protein A and monospecific antibodies to the CH2 domain and hinge region).

In Vitro Refolding of Cyclomaltodextrin Glucanotransferase from Cytoplasmic Inclusion Bodies Formed upon Expression in Escherichia coli

The recovery of active cyclomaltodextrin glucanotransferase (CGT) from inclusion bodies formed upon expression of a signal sequence deletion mutant ( cgtΔ ss) gene in Escherichia coli was studied. Under the conditions examined the in vitro renaturation yield of active enzyme was up to 81% of the maximum activity from urea solubilized and up to 3.7% from guanidinium hydrochloride (GdmCl) solubilized inclusion bodies. Refolding from GdmCl solutions resulted in the reaggregation of ΔssCGT. Although native (folded) CGT showed enzymatic activity at urea concentrations as high as 10 M, the inclusion bodies could be solubilized with 3 M urea solution, but the preparation had no enzymatic activity prior to reduction of the denaturant concentration. This suggests that the ΔssCGT inclusion bodies are composed of molecules that are trapped in an inactive state. The individual molecules may have extensive secondary structure, since inclusion bodies solubilized with 4.5 M urea gave maximum yield of activity in the renaturation step. With higher urea concentration the yield decreased. Thus, a "mild" solubilization technique seems to increase the yield of active ΔssCGT from the inclusion bodies. The stability of the refolded ΔssCGT was identical to that of extracellularly produced wild type CGT, whereas a disulfide bond mutant, Ser-70-CGT, showed reduced stability under identical conditions. This suggests that the single disulfide bond in ΔssCGT is formed during refolding and contributes to the stability of the molecule. A fusion of maltose binding protein to the NH 2-terminus of ΔssCGT did not significantly affect the in vitro folding of the ΔssCGT portion when tested by a denaturation-renaturation experiment.

Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium.

The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin. Analysis of observed chemical shifts and patterns of NOEs on the main chain indicates extensive and regular secondary structure throughout the N-terminal domain. In contrast, the helices of the C-terminal domain are somewhat irregular and are dynamically averaged. The EF-hands are intact in the N-terminal domain with the loops forming a short antiparallel beta sheet. Under low-salt conditions, two helix-loop-helix EF-hand motifs are present in the C-terminal domain of apoCaM but do not show interstrand NOEs. The spectral perturbations of apoCaM upon complexation with the Neurop peptide are relatively small with the larger chemical shift perturbations occurring in the C-terminal domain. The general secondary structure and tertiary organization appears to remain roughly the same as in free apoCaM. Stoichiometric titration of the apoCaM.Neurop complex with calcium indicates that the C-terminal domain EF-hands have a higher affinity for calcium than N-terminal domain EF-hands. Thus, this complex offers a unique opportunity to examine the structural and energetic consequences of calcium-dependent and calcium-independent binding of peptide to calmodulin.

Ornithine decarboxylase as a target for chemoprevention.

l-Ornithine decarboxylase (ODC) is essential for polyamine synthesis and growth in mammalian cells; it provides putrescine that is usually converted into the higher polyamines, spermidine and spermine. Many highly specific and potent inhibitors of ODC are based on the lead compound alpha-difluoromethylornithine (DFMO), which is an enzyme-activated irreversible inhibitor. DFMO is accepted as a substrate by ODC and is decarboxylated, leading to the formation of a highly reactive species that forms a covalent adduct with either cysteine-360 (90%) or lysine-69 (10%). Both modifications inactivate the enzyme. ODC activity is normally very highly regulated at both transcriptional and post-transcriptional levels according to the growth state of the cell and the intracellular polyamine content. Experimental over-production of ODC can be caused by either transfection with plasmids containing the ODC cDNA with part of the 5'-untranslated region (5'UTR) deleted under the control of a very strong viral promoter, or transfection of plasmids that cause the overproduction of eIF-4E, reported to be a limiting factor in the translation of mRNAs with extensive secondary structures in the 5'UTR. In both cases, unregulated overexpression of ODC transforms NIH 3T3 cells to a neoplastic state. Along with studies showing that many tumor promoters increase ODC activity and that a number of preneoplastic conditions and tumor samples show high levels of ODC, these results suggest that ODC may act as an oncogene in an appropriate background. This provides a rationale for the possible use of ODC inhibitors as chemopreventive agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Nrs1, a repetitive element linked to pisatin demethylase genes on a dispensable chromosome of Nectria haematococca.

We have identified a repetitive DNA element in Nectria haematococca mating population VI, isolate T-2. This repetitive sequence has been called Nrs1. DNA hybridization analysis indicates the sequence is found in several isolates of the fungus pathogenic to Pisum sativum. A 2,027-bp clone containing the Nrs1-2 allele contains a long polyA sequence, imperfect RNA polymerase III promoter sequences, multiple inverted repeats, and the potential for extensive secondary structure similar to known RNA polymerase III transcripts and related retroelements. Ten of the 11 HindIII restriction fragments from isolate T-2 DNA that hybridize to Nrs1-2 segregate in a manner consistent with a 1:1 ratio for random ascospore progeny. The 10 restriction fragment length polymorphism (RFLP) loci define three linkage groups and correspond to three chromosome-sized DNAs from T-2 separated by pulsed field gel electrophoresis. Three RFLP loci defined by hybridization to the gene for pisatin demethylase and localized on the 1.6 million base pair (Mb) chromosome were genetically linked to each other and to several Nrs1 loci. These sequences recombined despite the fact that no obvious homolog exists for the 1.6-Mb chromosome in one parent strain. Allelic RFLPs corresponding to the gene sequence of cutinase were unlinked to Nrs1 loci.

Human immunodeficiency virus nucleocapsid protein accelerates strand transfer of the terminally redundant sequences involved in reverse transcription.

During the initial stages of human immunodeficiency virus (HIV) replication, 5'-terminally redundant (R') DNA, the minus strand synthesized as the complement of the 5'-long terminal repeat (LTR) terminal redundancy, must anneal to the 3'-LTR RNA to enable template transfer. The (R')DNA sequences contain the site involved in the tat-TAR interaction and extensive secondary structures that strongly interfere with annealing. The novel annealing reaction between (R')DNA and 3'-LTR RNA follows first-order kinetics, consistent with an unusually slow unfolding of the secondary structure as the rate-limiting step followed by a more rapid nucleation step. The HIV nucleocapsid protein accelerates the annealing reaction 3000-fold under optimal conditions. This acceleration may be necessary for strand transfer to efficiently occur in vivo and may provide a target for anti-HIV chemotherapeutic agents.

Structural Characteristics of the Stable RNA Introns of Archaeal Hyperthermophiles and their Splicing Junctions

Five pre-23 S rRNA introns have been characterized for hyperthermophilic archaea. The small ones constitute stable core structures while the three larger ones contain, in addition, open reading frames, two of which encode homing-type endonucleases. The higher order structures of in vitro trancripts of the introns and their exon-intron junctions were examined using chemical and ribonuclease probes specific for unpaired nucleotides, double helical regions and helIx-loop junctions. The experimental data support both the formation of a "bulge-helix-bulge" structural motif at the exon-intron junctions, which is recognized by a cleavage enzyme, and the presence of an adjacent core structure consisting of a long, stable, stem-loop structure. Moreover, the data were used to derive secondary structural models for the three large introns which form stable, circular, RNA species, in vivo. Each exhibits extensive secondary structure and additional, as yet undefined, higher order structure, compatible with their high level of stability in vivo; limited structural similarities were detected between the two introns encoding the homing-type endonucleases. The data are compatible with the hypothesis that RNA introns can be transmitted between archaeal hyperthermophile cells.

Role of the 5'-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine.

S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5'-untranslated region (5'UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5'UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5'UTR, as well as chimaeric constructs in which the AdoMetDC 5'UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5'UTR, the AdoMetDC protein-coding region and the 3'UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5'UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5'UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5' end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5'UTR that may include but is not entirely due to the internal ORF.

The Origins of Protein Secondary Structure: Effects of Packing Density and Hydrogen Bonding Studied by a Fast Conformational Search

Globular proteins fold to create compact structures rich in α-helices and β-sheets. While studies of cubic lattice models of simplified polypeptide chains have concluded that, secondary structure is a necessary consequence of chain compactness, different conclusions have been reached from studies of off-lattice models of simplified chains. In an attempt to resolve this controversy, we study an all-atom off-lattice model of a protein subject to a variety of simplified energy functions. A Monte Carlo simulated annealing algorithm is used to search conformational space quickly. The algorithm uses pivot-type moves in which a residue is selected at random and the values of its main-chain dihedral angles are changed. The energy function used to accept or reject moves is taken to be either a term proportional to the volume occupied by a structure (to mimic the hydrophobic effect), a term proportional to the energy of main-chain hydrogen bonding, or a combination of these two terms. Secondary structure content is evaluated using several different definitions. For all the definitions used, compactness alone produces a 10% increase in secondary structure content. However, this is a small fraction of the secondary structure observed in native protein structures. Structures produced by minimizing the hydrogen bond energy have extensive secondary structure but are not densely packed. Structures having both the high density of native structures and extensive secondary structure are produced by minimizing combinations of the volume and hydrogen bond energy terms.Our results emphasize the close relationship between secondary structure and the geometry of main-chain hydrogen bonding. The results are consistent with a description of protein folding in which the hydrophobic effect favors dense packing while hydrogen bonding determines the specific local geometry which generates secondary structure. To make an analogy with lattice studies of packing density and secondary structure, it seems that hydrophobicity provides the packing density while hydrogen bonding provides the lattice.

Cloning and characterization of ERF-1, a human member of the Tis11 family of early-response genes.

Members of the Tis11 family of early-response genes are characterized by a high degree of sequence similarity around a putative zinc finger motif. They are induced by a variety of cell agonists and polypeptide mitogens, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF). We describe the cloning and sequencing of a human member of this gene family, EGF-response factor 1 (ERF-1), the homolog of the mouse Tis11b/rat cMG1 genes. The human and rodent genes are similar, with 5' UTR, coding sequence, and 3' UTR highly conserved. The promoter/enhancer region and intron sequences contain multiple putative transcription factor binding motifs characteristic of early-response genes. Amino acid sequence comparison of the seven members of the Tis11 family cloned so far identifies a repeated consensus motif of (x+)YKTELC(x+)x5GxCxYGx(x+)CxFxH involving the potential zinc finger. Toward the carboxyterminal end is a region with a high percentage of prolines (15/73) and, partially overlapping, a serine-rich domain (20/54). These may be important as trans-activation and phosphorylation sites. The 3' untranslated region is unusually long, extending over 1,860 bp. The sequence immediately downstream from the translational stop codon has extensive secondary structure potential. The 3' UTR is 60% AT rich, but contains two GC rich (> 70%) regions. In addition there are multiple reiterations of a destabilization sequence, as well as a single UUAUUUAU motif characteristic of mRNAs specifying proteins involved in the inflammatory response. The mRNA contains a consensus polyadenylation signal.

Trans complementation by RNA of defective foot-and-mouth disease virus internal ribosome entry site elements

A region of about 435 bases from the 5' noncoding region of foot-and-mouth disease virus RNA directs internal initiation of protein synthesis. This region, termed the internal ribosome entry site (IRES), is predicted to contain extensive secondary structure. Precise deletion of five predicted secondary structure features has been performed. The mutant IRES elements have been constructed into vectors which express bicistronic mRNAs and assayed within cells. Each of the modified IRES elements was defective in directing internal initiation when assayed alone. However, coexpression of an intact foot-and-mouth disease virus IRES complemented four of these defective elements to an efficiency of up to 80% of wild-type activity. No complementation was observed with the structurally analogous element from encephalomyocarditis virus. The role of RNA-RNA interactions in the function of the picornavirus IRES is discussed.

CDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor

We isolated five independent cDNAs of nearly 3000 bp for the bovine ACTH receptor by screening adrenal cortex cDNA libraries with a PCR cloned cDNA fragment. The deduced receptor sequence includes 297 residues ( M r = 33 258) with 81% identity with the human ACTH receptor, and shows seven hydrophobic transmembrane domains. The calculated M r of the receptor is smaller than the 40–45 kDa observed in crosslinking studies with labeled ACTH. Since the bovine and human receptors have two glycosylation motifs in the N-terminus, the difference may result from glycosylation of the receptor. Analysis of the sequences of both bovine and human receptors revealed a single protein kinase A phosphorylation motif located in the third intraceullular loop (Ser-209) juxtaposed to a protein kinase C phosphorylation motif (Thr-204). Thus, the involvement of protein kinase A and C pathways in ACTH action may be mediated in part by phosphorylation of the ACTH receptor at these motifs. The 3′-untranslated region of the bovine cDNA is > 2000 bp and includes two inverse repeats giving an extensive and strong secondary structure to the ACTH receptor RNA.

Secondary structure characterization of ß-lactamase inclusion bodies

The secondary structure of proteins in E. coli inclusion bodies was investigated via Raman spectroscopy. Inclusion bodies were purified from cells expressing different forms of RTEM beta-lactamase and grown at either 37 or 42 degrees C. All of the solid phase inclusion body samples examined gave amide I band spectra that were perturbed from that of the native, purified protein in both solution and powder forms; secondary structure estimates indicated significant decreases in alpha-helix and increases in beta-sheet contents in the inclusion body samples. The structure estimates for inclusion bodies isolated from 37 degrees C cultures were similar, regardless of aggregate localization in the E. coli cytoplasmic or periplasmic spaces or beta-lactamase precursor content. Inclusion bodies obtained from 42 degrees C cells exhibited a further reduction of alpha-helix and augmentation of beta-sheet contents relative to those from 37 degrees C cultures. These results are consistent with the paradigm for inclusion body formation via the self-association of intra-cellular folding intermediates having extensive secondary structure content. Further, the overall secondary structure content of inclusion bodies is not significantly affected by subcellular compartmentalization, but may be altered at increased temperatures.

Partially folded states of equine lysozyme. Structural characterization and significance for protein folding.

Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly dependent on the state of calcium binding. Differential scanning calorimetry shows the two transitions to be particularly well resolved in the calcium-free protein, where the first transition occurs with a midpoint at 44 degrees C at pH 4.5 or in 0.8 M GdnHCl at pH 7.5, 25 degrees C, and the second occurs near 70 degrees C at pH 4.5 or in 3.7 M GdnHCl at pH 7.5, 25 degrees C. In the presence of calcium, the first transition takes place with a midpoint of 55 degrees C or in excess of 2.5 M GdnHCl, but the parameters for the second transition remain unchanged. Fluorescence emission and UV difference absorption spectroscopy suggest that the first transition generates an intermediate state in which sequestration of some aromatic side chains from solvent has occurred whereas the second represents denaturation to a highly unfolded state. CD and 1H NMR results indicate that the intermediate state possesses extensive secondary and tertiary structure, although the latter is substantially disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

Refolding of brain-derived neurotrophic factor from guanidine hydrochloride: Kinetic trapping in a collapsed form which is incompetent for dimerization

We have studied the pathway and kinetics of refolding of recombinant human brain-derived neurotrophic factor (BDNF), which is a very tightly-associated dimer in its native state. When BDNF unfolded in 6 M guanidine hydrochloride is diluted 20-fold into phosphate-buffered saline, a partially folded intermediate is rapidly formed (< 1 min). Circular dichroism and fluorescence spectroscopy show that this intermediate has extensive secondary structure, but no well-defined tertiary structure. Size-exclusion chromatography with light scattering detection shows that it is compact and monomeric, and therefore corresponds to what is often called a "collapsed form" or "molten globule". This collapsed form disappears with a half-time of approximately 30 min, simultaneously with the appearance of native dimers, without accumulation of monomeric species with a native tertiary structure. Remarkably, the monomer-dimer association constant of the collapsed form is approximately 10(10) weaker than the native structure, and it has a low tendency to form large aggregates. Given the very large hydrophobic surface present at the dimer interface of nerve growth factor (and presumably in BDNF), these results indicate that these hydrophobic groups are not exposed in the collapsed form, and that it is therefore quite dissimilar from the native structure. A significant conformational change in the collapsed form is necessary to re-expose these hydrophobic groups to form the dimer interface, making this the rate-limiting step in reaching the native conformation.