Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation.

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.     

Structural analysis based on state-space modeling.

A new method has been developed to compute the probability that each amino acid in a protein sequence is in a particular secondary structural element. Each of these probabilities is computed using the entire sequence and a set of predefined structural class models. This set of structural classes is patterned after Jane Richardson''s taxonomy for the domains of globular proteins. For each structural class considered, a mathematical model is constructed to represent constraints on the pattern of secondary structural elements characteristic of that class. These are stochastic models having discrete state spaces (referred to as hidden Markov models by researchers in signal processing and automatic speech recognition). Each model is a mathematical generator of amino acid sequences; the sequence under consideration is modeled as having been generated by one model in the set of candidates. The probability that each model generated the given sequence is computed using a filtering algorithm. The protein is then classified as belonging to the structural class having the most probable model. The secondary structure of the sequence is then analyzed using a "smoothing" algorithm that is optimal for that structural class model. For each residue position in the sequence, the smoother computes the probability that the residue is contained within each of the defined secondary structural elements of the model. This method has two important advantages: (1) the probability of each residue being in each of the modeled secondary structural elements is computed using the totality of the amino acid sequence, and (2) these probabilities are consistent with prior knowledge of realizable domain folds as encoded in each model. As an example of the method''s utility, we present its application to flavodoxin, a prototypical alpha/beta protein having a central beta-sheet, and to thioredoxin, which belongs to a similar structural class but shares no significant sequence similarity.     

Organization of the genes necessary for hydrogenase expression in Rhodobacter capsulatus. Sequence analysis and identification of two hyp regulatory mutants

A 25kbp DNA fragment from the chromosome of Rhodobacter capsulatus B10 carrying hydrogenase (hup) determinants was completely sequenced. Coding regions corresponding to 20 open reading frames were identified. The R. capsulatus hydrogenase-specific gene (hup and hyp) products bear significant structural identity to hydrogenase gene products from Escherichia coli (13), from Rhizobium liguminosarum (16), from Azotobacter vinelandii (10) and from Alcaligenes eutrophus (11). The sequential arrangement of the R. capsulatus genes is: hupR2-hupU-hypF -hupS-hupL-hupM-hupD -hupF -hupG -hupH -huoJ -hupK -hypA-hypB-hupR1-hypC -hypD -hypE -ORF19 -ORF20 , all contiguous and transcribed from the same DNA strand. The last two potential genes do not encode products that are related to identified hydrogenase-specific gene products in other species. The sequence of the 12 R. capsulatus genes underlined above is presented. The mutation site in two of the Hup^? mutants used in this study, RS13 and RCC12, was identified in the hypF gene (deletion of one G) and in the hypD qene (deletion of 54 bp), respectively. The hypF gene product shares 45% identity with the product of hydA from E. coli and the product of hypF from R. leguminosarum. Those products present at their N-terminus a Cys arrangement typical of zinc-finger proteins. The G deletion in the C-terminal region of hypF in the RS13 mutant     

Peptide selection by class I molecules of the major histocompatibility complex

Class I molecules of the major histocompatibility complex (MHC) bind peptides derived from cytoplasmic proteins. Comparison of over 100 such peptides reveals the importance of the carboxy-terminal residue in selective binding. Recent evidence implicates the proteases and transporters of the processing pathway in providing peptides with the correct residues at the carboxyl terminus.     

Genistein inhibits calcium release by platelet-derived growth factor but not bradykinin or cadmium in human fibroblasts

Cd²⁺ provokes inositol trisphosphateproduction and releases stored Ca²⁺, apparently by binding to a zinc site in the external domain of an orphan receptor. One pM Cd²⁺ evokes an immediate spike in cytosolic free Ca²⁺, which is similar to that evoked by bradykinin. Platelet-derived growth factor (PDGF) also increases free Ca²⁺ in human dermalfibroblasts, but there is a distinct lag before free Ca²⁺ rises in response to PDGF. Genistein, which selectively inhibits tyrosine kinases, markedly inhibited Ca²⁺ mobilization evoked by PDGF. Calcium mobilization triggered by cadmium or bradykinin was relatively insensitive to genistein. The PDGF receptor is known to be a tyrosine kinase, whichphosphorylates and thereby activatesphospholipase C, whereas a G protein couples the bradykinin receptor to anotherphospholipase C isoform. These findings support the hypothesis that the orphan receptor triggered by cadmium is coupled to phospholipase C via a G protein.Abbreviations BSA bovine serum albumin - BK bradykinin - [Ca²⁺]_i cytosolic free calcium - DME Dulbecco's modified Eagle's medium - FBS fetal bovine serum - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - IC₅₀ concentration that produces 50% inhibition - PDGF platelet-derived growth factor - PSS physiological salts solution - SE standard error of the mean