Temperature-jump NMR study of protein folding: Ribonuclease A at low pH

Summary The kinetic process of folding of bovine pancreatic ribonuclease A in a²H₂O environment at pH 1.2 was examined by a recently developed temperature-jump NMR method (Akasaka et al., (1990) Rev. Sci. Instrum.61, 66–68). Upon temperature-jump down from 45°C to 29°C, which was attained within 6 s, the proton NMR spectral changes were followed consecutively in time intervals of seconds. There was a rapid spectral change, which was finished within the jump period, followed by a much slower process which lasted for a minute or longer. Rates of the slower process were measured at different positions of the polypeptide chain as intensity changes of individual His and Tyr proton signals of the folded conformer and as intensity changes of aliphatic and His protons of the unfolded conformer. Most of these rates coincided with each other within experimental error with an average value of 2.8×10^–2s^–1. The result gave clear experimental evidence that the slow folding of RNase A at low pH is a cooperative process involving most regions of the molecule, not only thermodynamically, but kinetically as well.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Structure-based functional inference in structural genomics

The dramatically increasing number of new protein sequences arising from genomics 4 proteomics requires the need for methods to rapidly and reliably infer the molecular and cellular functions of these proteins. One such approach, structural genomics, aims to delineate the total repertoire of protein folds in nature, thereby providing three-dimensional folding patterns for all proteins and to infer molecular functions of the proteins based on the combined information of structures and sequences. The goal of obtaining protein structures on a genomic scale has motivated the development of high throughput technologies and protocols for macromolecular structure determination that have begun to produce structures at a greater rate than previously possible. These new structures have revealed many unexpected functional inferences and evolutionary relationships that were hidden at the sequence level. Here, we present samples of structures determined at Berkeley Structural Genomics Center and collaborators laboratories to illustrate how structural information provides and complements sequence information to deduce the functional inferences of proteins with unknown molecular functions.Two of the major premises of structural genomics are to discover a complete repertoire of protein folds in nature and to find molecular functions of the proteins whose functions are not predicted from sequence comparison alone. To achieve these objectives on a genomic scale, new methods, protocols, and technologies need to be developed by multi-institutional collaborations worldwide. As part of this effort, the Protein Structure Initiative has been launched in the United States (PSI; www.nigms.nih.gov/funding/psi.html). Although infrastructure building and technology development are still the main focus of structural genomics programs [1–6], a considerable number of protein structures have already been produced, some of them coming directly out of semi-automated structure determination pipelines [6–10]. The Berkeley Structural Genomics Center (BSGC) has focused on the proteins of Mycoplasma or their homologues from other organisms as its structural genomics targets because of the minimal genome size of the Mycoplasmas as well as their relevance to human and animal pathogenicity (http://www.strgen.org). Here we present several protein examples encompassing a spectrum of functional inferences obtainable from their three-dimensional structures in five situations, where the inferences are new and testable, and are not predictable from protein sequence information alone.     

Protein Sequence Modules

Abstract

Conserved protein sequence segments are commonly believed to correspond to functional sites in the protein sequence. A novel approach is proposed to profile the changing degree of conservation along the protein sequence, by evaluating the occurrence frequencies of all short oligopeptides of the given sequence in a large proteome database. Thus, a protein sequence conservation profile can be plotted for every protein. The profile indicates where along the sequences the potential functional (conserved) sites are located. The corresponding oligopeptides belonging to the sites are very frequent across many prokaryotic species. Analysis of a representative set of such profiles reveals a common feature of all examined proteins: they consist of sequence modules represented by the peaks of conservation. Typical size of the modules (peak-to-peak distance) is 25–30 amino acid residues.     

Inhibitory effect of bFGF on endochondral heterotopic ossification

Basic fibroblast growth factor (bFGF) is reported to stimulate repair of fracture and bony defects in in vivo animal studies. However, most studies performed in vitro demonstrate inhibitory effect of bFGF on cartilage and bone differentiation. To understand the discrepancy observed in in vivo and in vitro studies, we evaluated the effect of bFGF on chondro-osteogenesis initiated by bone matrix powder (MP). MP was implanted in the murine hamstring muscles with or without administration of bFGF. Injection of 1 microg of bFGF markedly reduced the size of heterotopic bone induced by MP, as detected by X-ray. Injection of 10 microg of bFGF completely inhibited ossification and only fibrous tissues were observed at the site of MP implantation. The expressions of alkaline phosphatase and osteocalcin mRNAs, markers for bone differentiation, were completely suppressed by 10 microg of bFGF. These results demonstrate the inhibitory effect of bFGF on endochondral ossification in vivo, implicating a precaution for its use in musculo-skeletal disorders.     

Is the failing heart out of fuel or a worn engine running rich? A study of mitochondria in old spontaneously hypertensive rats

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause-and-effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20-month-old SHR and Wistar-Kyoto controls by iTRAQ-labelling combined with multidimensional LC/MS/MS. Of 137 high-scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Alpha repeat proteins (αRep) as expression and crystallization helpers

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.     

Capacity for protein synthesis following heat stimulus of Drosophila associates with heat tolerance but does not underlie the latitudinal tolerance cline

Across populations of Drosophila melanogaster along the Australian eastern coastline latitudinal clines occur in both heat-knockdown tolerance and hardened heat-knockdown tolerance – low latitude tropical populations being more tolerant. A latitudinal cline also occurs for rates of total protein synthesis following a mild heat stress, with tropical populations having higher rates. Since the control of protein synthesis following heat stress is an important component of the cellular heat-shock response, we hypothesised that the higher rates of synthesis that follow a heat stimulus lead to higher knockdown tolerance and underpins the cline. However, levels of heat-stimulated total protein synthesis have been negatively related to heat-hardening capacity, a somewhat conflicting result. Here we examine the relationship between these physiological and adaptive traits in a set of 40 family lines derived from a hybrid laboratory population established by crossing populations from either end of the latitudinal transect. Among these lines high levels of heat-stimulated total protein synthesis were associated with both low basal and low heat-hardened adult knockdown time, confirming the importance of a negative relationship between protein synthesis and thermal tolerance. This result, when considered along with the directions of the latitudinal clines in protein synthesis and tolerance, suggests that variation in rates of heat-stimulated total protein synthesis is not a factor contributing to the latitudinal cline in heat tolerance. Given the robustness of this negative relationship we discuss possible explanations and future experiments to elucidate how the cellular heat stress response might facilitate increased knockdown tolerance.