Association between Polymorphisms of Heat Shock Protein HSPA5 and Risk of Type 2 Diabetes Mellitus.

We studied the relationship between the HSPA5 gene polymorphisms and the risk of type 2 diabetes mellitus. Genotyping of three SNPs of the HSPA5 gene was performed in 1579 patients with type 2 diabetes mellitus and 1650 healthy individuals. It was found that the genotypes rs55736103-T/T, rs12009-G/G, and rs391957-T/C-T/T are associated with increased risk of type 2 diabetes in females. A rare haplotype, rs55736103C-rs12009A-rs391957T HSPA5, associated with a reduced risk of type 2 diabetes in females was found. Associations between polymorphisms of the HSPA5 gene encoding heat shock protein and the risk of type 2 diabetes mellitus were established for the first time.

[Comparative analysis of the intestinal microbiota in patients with exocrine pancreatic insufficiency of various severity].

BACKGROUND: Exocrine pancreatic insufficiency (EPI) is a critical host factor in determining the composition of the gut microbiota. Diseases that cause exocrine insufficiency can affect the gut microbiome, which can potentiate disease progression and complications. To date, the relationship of exocrine insufficiency in various pancreatic (PA) pathologies, in chronic pancreatitis (CP), with dysbiotic changes in the intestinal microbiota (IM) has not been reliably studied. The available data are heterogeneous and contradictory, which determines the need for further research. AIM: To conduct a comparative analysis of the taxonomic composition of the intestinal microbiota in patients with CP of various etiologies, without or with the presence of EPI of varying severity, as well as patients with severe EPI with a history of surgical intervention (SI) on the pancreas. MATERIALS AND METHODS: A total of 85 patients were included in the study. Patients were divided into groups according to the severity of EPI: Group 1 (n=16) - patients with CP without EPI; Group 2 (n=11) - patients with CP and mild EPI; Group 3 (n=17) - patients with severe CP and EPI; Group 4 (n=41) - severe EPI in persons with a history of SI on the pancreas. Verification of CP was carried out according to clinical, anamnestic and instrumental data. The degree of EPI was determined by the level of pancreatic elastase-1 (PE-1) feces. Informed consent for the study was obtained for each patient, an anamnesis was collected, physical and laboratory examinations were performed, and a stool sample was obtained. DNA was extracted from each stool sample, the taxonomic composition of BM was determined by sequencing the bacterial 16S rRNA genes, followed by bioinformatic analysis. RESULTS: We followed the changes in the gut microbiota from a group of patients with CP without EPI to a group with severe EPI, in those who underwent SI. At the level of the phylum, the IM of all groups showed the dominance of Firmicutes, with the lowest representation in the severe EPI group, both with SI and CP, and the growth of the Actinobacteria, Verrucomicrobiota and Fusobacteria types. The differential representation of childbirth varied: in patients with severe EPI and CP, compared with mild, statistically significant genera - Akkermansia, Ruminococcus gauvreauii group and Holdemanella; compared with CP without exocrine insufficiency, Prevotella, Ruminococcus gauvreauii group, Peptostreptococcus and Blautia dominated. The CP group with mild EPI was dominated by the following genera: Lachnospiraceae_ND 2004 group, Faecalitalea, Fusobacterium, Catenibacterium, Roseburia, Atopobium, Cloacibacillus, Clostridium innococum group, Ruminococcus torques group. All groups showed a low diversity of taxa with a predominance of opportunistic flora, including participants in oncogenesis. CONCLUSION: The results of the study show that patients with CP of various etiologies and patients with severe EPI who underwent specific intervention on the pancreas have intestinal microbiota dysbiosis, the severity of which is significantly influenced by the degree of EPI.

[Polypragmasia and interdrug interactions as risk factors of falling in elderly patients.]

This article presents the results of the analysis of data from patients over 75 years of age from a multidisciplinary hospital with cardiovascular disease and comorbid conditions. Pharmacotherapy of gerontological patients with multiple risk factors for falls was analysed in terms of the presence of polypragmasy and drug-drug interactions hazardous to the risk of falls. In the group of patients who experienced a fall in hospital compared to patients without a fall, the prescription lists audit showed a predominance of medicines (drugs) and drug combinations compromised by an increased risk of this serious adverse event. An audit of prescriptions of patients at increased risk of falls as a means of combating polypharmacy and identifying drugs that may cause falls can be conducted using the «Traffic light classification of FRIDs¼ and drug checkers to identify clinically relevant combinations. The use of these clinical and pharmacological tools can improve the quality and safety of medical care in a hospital setting.

[Polypragmasy: A clinical pharmacologist's view]. / Polipragmaziya: vzglyad klinicheskogo farmakologa.

In the modern world, there is a rapid advance in the design and clinical introduction of a huge number of drugs that are able to cure a patient or to improve his/her health status on the one hand and to cause significant harm to his/her health on the other. Polypragmasy is the desire to enhance the efficiency of treatment and to help the patient recover from all developed diseases inevitably leads to the use of a large number of medications. At the present time, polypragmasy as a result of iatrogenia is a serious public health problem, as it is clinically manifested by a reduction in the effectiveness of pharmacotherapy, by the development of severe adverse drug reactions, and by a considerable increase in healthcare expenditures. The reason for the simultaneous prescription of multiple drugs may be comorbidity (multimorbidity), the availability of drugs, as well as clinical guidelines, manuals of professional medical associations, treatment standards that contain recommendations for using combination therapy with more than 5 drugs for only one disease in some cases, the efficiency of which corresponds to a high level of evidence. Currently, the fight against polypragmasy is one of the important tasks in rendering medical care to elderly and senile patients since it is a major risk factor of adverse drug reactions in this category of people. To minimize polypragmasy in elderly patients, it is necessary to use current methods for analyzing each prescription of a drug (the index of rational drug prescribing; an anticholinergic burden scale) and those for optimizing pharmacotherapy with the use of restrictive lists (Beers criteria, STOPP/START criteria) that will be able to reduce the number of errors in the administration of drugs and to maximize the efficiency and safety of pharmacotherapy.

[Discovery of Genetic Markers of Resistance to ß-Lactams in Gramnegative Microorganisms by PCR Diagnosis].

Fifteen specimens of the hemoculture and 89 specimens of the broncho-alveolar lavage were used in the study. Monocultures of gramnegative bacteria resistant to cefotaxime, cefepime, imipenem and meropenem were isolated from the specimens. The PCR method with detection of the results in the real time regimen (PCR test-system Litekh) provided detection of the beta-lactamase genes: bla(CTX-M-like) (72/104, 69.2%), bla(NDM) (6/104, 5.8%), bla(VIM) (49/104, 47.1%) and bla(OXA48-like) (59/104, 56.7%). There was identified correlation between the phenotype of resistance of Acinetobacter spp., Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli to cefotaxime and carbopenems and detection of the bla(CTX-M-like) and bla(NDM) genes. At the same time, up to 70% of the K. pneumoniae isolates from the biological specimes positive with respect to the presence of the carbapenase bla(VIM) and bla(OXA48-like) genes demonstrated their phenotypic susceptibility to carbopenems. The results of the study confirmed the prognostic value of the genetic diagnosis for improvement of the routine bacteriological investigations.

[Genetic determinants of antibiotic resistance in oropharyngeal streptococci in patients with chronic obstructive pulmonary disease and in those with asthma]. / Geneticheskie determinanty ustoichivosti orofaringeal'nykh streptokokkov k antibakterial'nym sredstvam u patsientov s khronicheskoi obstruktivnoi bolezn'yu legkikh i bol'nykh bronkhial'noi astmoi.

AIM: To identify oropharyngeal Streptococcus species and to analyze the genetic determinants of antibiotic resistance in patients with asthma and in those with chronic obstructive pulmonary disease (COPD). MATERIAL AND METHODS: An experimental diagnostic Streptopol+ (Lytech Co. LTD) panel based on a multiplex real-time PCR was applied to investigate the representation of antimicrobial resistance genes (mef and ermB) and the species composition of streptococci isolated from oropharyngeal swab samples from 89 patients with stable COPD and from 51 patients with asthma. RESULTS: In the stable disease period, the oropharyngeal swabs were found to contain Streptococcus pneumoniae in 7.8% of the patients with asthma and in 6.74% of those with COPD; the common feature of these groups was a tendency towards a severe disease course and recurrent exacerbations requiring antibiotics. S. pyogenus was detected in 42.9% of the oropharyngeal swabs from COPD and asthma patients without exacerbations. The oropharyngeal swabs showed the mef gene in 100% of the patients with asthma and in 100% of those with COPD; the ermB gene was encountered in 91% of the patients with COPD and in 82.4% of those with asthma. The COPD patients displayed a direct correlation between the representation of the ermB gene and sputum production and smoking index. The mef and ermB genes were directly correlated with the frequency of exacerbations in patients with COPD. CONCLUSION: The identified streptococci are a reservoir of antimicrobial resistance genetic determinants - the mef and ermB genes encoding the mechanisms of streptococcal macrolide resistance. The representation of the above genes directly correlates with the frequency of exacerbations and the number of antimicrobial drug uses.

[Specific features of the enteric microbiota composition in patients with alcoholic liver cirrhosis].

AIM: To establish the specific features of the taxonomic and functional composition of the enteric microbiota in patients with alcoholic liver cirrhosis (LC). SUBJECTS AND METHODS: Metagenomic analysis was used to study the taxonomic composition and functional potential of the enteric microbiota in 20 patients with alcoholic LC. Total DNA was isolated from the patients' fecal samples; thereafter full genome sequencing was carried out. The metagenomic analysis yielded the results of the relative taxonomic and functional abundance of microbial species in the test samples. These were comparatively analyzed with the previously published metagenomic datasets of healthy population cohorts in the Russian Federation, as well as in Denmark, China, and the USA. RESULTS: In the majority of patients, the dominant part of the intestinal community represented bacterial species constituting the normal human intestinal flora. At the same time, abnormal gut microbiota composition, which was suggestive of marked dysbacteriosis, was identified in a number of patients. In addition, pooled analysis of the data could identify a number of species with a statistically significantly increase and decrease in the relative abundance as compared to the control groups. Thus, the enteric microbiota of the patients with alcoholic LC showed a high proportion of bacteria characteristic of the oral cavity. Analysis of the pooled metabolic potential of the microbiota in these patients demonstrated the higher abundance of enzyme genes involved in alcohol metabolism. CONCLUSION: In the patients with alcoholic LC, the microbiota composition changes identified in individual bacterial species may be associated with gastrointestinal comorbidities, such as chronic erosive gastritis, chronic pancreatitis, and gastric ulcer. The alterations occurring in alcoholic cirrhosis promote the penetration and generation of oral cavity-specific microorganisms in the human intestine. This may a potential biomarker for the diagnosis of liver diseases. The bacterial enzyme genes involved in alcohol metabolism have an increased abundance in patients with alcoholic LC and healthy volunteers from the Russian Federation.

[Psychological development of children with opsoclonus myoclonus syndrome and its correlation with age and disease onset]. / Psikhicheskoe razvitie detei s opsoklonus-mioklonus-sindromom s uchetom razlichii v vozraste, vremeni debiuta i sroka techeniia zabolevaniia.

Opsoclonus-myoclonus syndrome (OMS) is a rare neurological disorder with unknown etiology and pathogenesis. There is not enough knowledge about psychological development of children with OMS. AIM: To study an influence of the time of disease onset and children's age and type of disease course on psychological development of children with OMS. MATERIAL AND METHODS: The study included 26 children with OMS of age between 1 year 7 months and 13 years, 10 boys and 16 girls. The following methods were used: analysis of patient's development records, psychological interview with parents, assessment of emotional, behavioral, motion state and interpersonal relations. RESULTS AND CONCLUSION: The disease onset at the early stage of ontogenesis (before 3 years) results into severe psychological disorders. Patients at the age between 3 and 5 years demonstrate the maximal number of developmental delays with possibilities of compensation. The article discusses a possible role of psychotraumatic and psychosocial factors in psychological disorders in children with OMS. The influence of age should be taken into account in planning of treatment and rehabilitation and evaluation of disease prognosis.

Designed beta-sheet-forming peptide 33mers with potent human bactericidal/permeability increasing protein-like bactericidal and endotoxin neutralizing activities.

Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI. A library of 24 peptide 33mers (betapep-1 to betapep-24) were synthesized with various amino acid substitutions. CD and NMR data acquired in aqueous solution indicate that betapep peptides form beta-sheet structure to varying degrees and self-associate as dimers and tetramers like the alpha-chemokines. Bactericidal activity toward Gram-negative Pseudomonas aeruginosa was tested, and betapep-19 was found to be only about 5-fold less potent (62% kill at 1.2 x 10(-7) M) than native B/PI (80% kill at 2.9 x 10(-8) M). At LPS neutralization, betapep-2 and -23 were found to be most active (66-78% effective at 1.2 x 10(-6) M), being only about 50-100-fold less active than B/PI (50% at 1.5 x 10(-8) M). In terms of structure-activity relations, beta-sheet structural stability correlates with the capacity to neutralize LPS, but not with bactericidal activity. Although a net positive charge is necessary for activity, it is not sufficient for optimal activity. Hydrophobic residues tend to influence activities indirectly by affecting structural stability. Furthermore, results show that sequentially and spatially related residues from the beta-sheet domain of native B/PI can be designed into short linear peptides which show good beta-sheet folding and retain much of the native activity. This research contributes to the development of solutions to the problem of multiple drug-resistant, opportunistic microorganisms like P. aeruginosa and of agents effective at neutralizing bacterial endotoxin.

A folding pathway for betapep-4 peptide 33mer: from unfolded monomers and beta-sheet sandwich dimers to well-structured tetramers.

It was recently reported that a de novo designed peptide 33mer, betapep-4, can form well-structured beta-sheet sandwich tetramers (Ilyina E, Roongta V, Mayo KH, 1997b, Biochemistry 36:5245-5250). For insight into the pathway of betapep-4 folding, the present study investigates the concentration dependence of betapep-4 self-association by using 1H-NMR pulsed-field gradient (PFG)-NMR diffusion measurements, and circular dichroism. Downfield chemically shifted alphaH resonances, found to arise only from the well-structured betapep-4 tetramer state, yield the fraction of tetramer within the oligomer equilibrium distribution. PFG-NMR-derived diffusion coefficients, D, provide a means for deriving the contribution of monomer and other oligomer states to this distribution. These data indicate that tetramer is the highest oligomer state formed, and that inclusion of monomer and dimer states in the oligomer distribution is sufficient to explain the concentration dependence of D values for betapep-4. Equilibrium constants calculated from these distributions [2.5 x 10(5) M(-1) for M-D and 1.2 x 10(4) M(-1) for D-T at 313 K] decrease only slightly, if at all, with decreasing temperature indicating a hydrophobically mediated, entropy-driven association/folding process. Conformational analyses using NMR and CD provide a picture where "random coil" monomers associate to form molten globule-like beta-sheet sandwich dimers that further associate and fold as well-structured tetramers. Betapep-4 folding is thermodynamically linked to self-association. As with folding of single-chain polypeptides, the final folding step to well-structured tetramer betapep-4 is rate limiting.

A recipe for designing water-soluble, beta-sheet-forming peptides.

Based on observations of solubility and folding properties of peptide 33-mers derived from the beta-sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-alpha), as well as other beta-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced beta-sheet-forming peptides. CD, 1H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like beta-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, native-like beta-sheet dimers at higher pH. Gro-alpha peptide is soluble at all pH values, yet displays no discernable beta-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble beta-sheet-forming peptides calls for the peptide to contain 40-50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form beta-sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact beta-sheet tetramer, whereas the others are transient beta-sheet-containing aggregates.

Backbone and side-chain dynamics of residues in a partially folded beta-sheet peptide from platelet factor-4.

Structurally characterizing partially folded states is problematic given the nature of these transient species. A peptide 20mer, T38AQLIATLKNGRKISLDLQA57 (P20), which has been shown to partially fold in a relatively stable turn/loop conformation (LKNGR) and transient beta-sheet structure, is a good model for studying backbone and side-chain mobilities in a transiently folded peptide by using 13C-NMR relaxation. Here, four residues in P20, A43, T44, G48, and 151, chosen for their positions in or near the loop conformation and for compositional variety, have been selectively 13C-enriched. Proton-coupled and decoupled 13C-NMR relaxation experiments have been performed to obtain the temperature dependencies (278 K to 343 K) of auto- and cross-correlation motional order parameters and correlation times. In order to differentiate sequence-neighbor effects from folding effects, two shorter peptides derived from P20, IATLK (P5) and NGRKIS (P6), were similarly 13C-enriched and investigated. For A43, T44, G48, and 151 residues in P20 relative to those in P5/P6, several observations are consistent with partial folding in P20: (1) C alpha H motional tendencies are all about the same, vary less with temperature, and are relatively more restricted, (2) G48 C alpha H2 phi (t) psi (t) rotations are more correlated, and (3) methyl group rotations are slower and yield lower activation energies consistent with formation of hydrophobic "pockets." In addition, T44 and 151 C beta H mobilities in P20 are more restricted at lower temperature than those of their C alpha H and display significantly greater sensitivity to temperature suggesting a larger enthalpic contribution to side-chain mobility. Moreover, at higher temperatures, side-chain methyls and methylenes in P20 are more motionally restricted than those in P5/P6, suggesting that some type of "folded" or "collapsed" structure remains in P20 for what normally would be considered an "unfolded" state.

Design of a potent novel endotoxin antagonist.

BACKGROUND: Bactericidal permeability increasing protein (BPI) binds to and neutralizes lipopolysaccharide (LPS, endotoxin). Small synthetic peptides based on the amino acid sequence of the LPS binding domain of BPI neutralize LPS, albeit inefficiently. Although the LPS binding domain of native BPI possesses a beta-turn secondary structure, this structure is not present in small derivative peptides. The purpose of this study was to determine whether the addition of a beta-turn to a BPI-derived peptide is associated with more potent endotoxin antagonism. METHODS: We generated a hybrid peptide (BU3) on the basis of (1) a portion of the LPS binding domain from BPI and (2) amino acids known to initiate a beta-turn. BU3 folds with a beta-turn, and we tested its effects on LPS neutralization and LPS-induced tumor necrosis factor-alpha secretion, comparing it with BPI-derived peptide BG22 that lacks a beta-turn and to an irrelevant peptide (BG16). RESULTS: Compared with BG22, BU3 demonstrated enhanced LPS neutralization and inhibition of LPS-induced tumor necrosis factor-alpha secretion in vitro and a similar diminution of endotoxemia and tumor necrosis factor-alpha secretion in a murine model of endotoxemia. CONCLUSIONS: These data demonstrate the potential for enhancing the biologic activity of a BPI-derived peptide endotoxin antagonist via manipulation of its conformational structure.

Anisotropic diffusion in etched particle tracks studied by field gradient NMR.

Etched particle tracks produced after heavy ion irradiation of polymer foils are used as model systems to test the performance of NMR in a newly developed ultrahigh magnetic field gradient system. The stimulated NMR echo decay of molecules diffusing in the channels, formulated in terms of the self part of the intermediate scattering function, is anisotropic and yields the form factor of the channels.

Trypsin-like enzyme from Streptomyces 771. Purification and properties of native and immobilized enzyme.

Electrophoretically homogeneous proteolytic enzyme with molecular weight 31,500 and pI 3.75 was obtained from a culture medium of Streptomyces 771 by chromatography on N-benzyl chitin adsorbent, subsequent chromatography on CM-cellulose, and preparative isofocusing and chromatography on Sephadex G-75. The enzyme hydrolyzes N-benzoyl-DL-arginine-p-nitroanilide N-benzoyl-DL-lysine-p-nitro-anilide N-benzoyl-DL-arginine ethyl ester, and Na-caseinate. It also exhibits pronounced thrombolytic activity. The activity of the enzyme was suppressed by soya bean inhibitor, but remained unaffected by chelating agents and phenylmethylsulfonyl fluoride. The enzyme was immobilized on aldehyde dextran, and some kinetic parameters of the immobilized enzyme were determined. The thrombolytic activity of native and immobilized enzyme was studied as well.

Multiple native-like conformations trapped via self-association-induced hydrophobic collapse of the 33-residue beta-sheet domain from platelet factor 4.

Native platelet factor 4 (PF4) (70 residues) has a hydrophobic three-stranded anti-parallel beta-sheet domain on to which is folded an amphipathic C-terminal alpha-helix and an aperiodic N-terminal domain. The 33-amino acid beta-sheet domain from PF4 (residues 23-55) has been synthesized and studied by c.d. and n.m.r. At 10 degrees C and low concentration, peptide 23-55 appears to exist in aqueous solution in a random-coil distribution of highly flexible conformational states. Some preferred conformation, however, is observed, particularly within a relatively stable chain reversal from Leu-45 to Arg-49. As the peptide concentration and/or temperature is increased, a new conformational state(s) appears and intensifies as slowly exchanging (600 MHz 1H-n.m.r. chemical-shift time scale) random-coil resonances disappear. Hill plots of the concentration-dependence indicated mostly tetramer formation as found in native PF4. Although apparent resonance linewidths in aggregate state(s) are of the order of 100 Hz, sequence-specific assignments for most resonances could be made. N.m.r./nuclear Overhauser effect structural analysis indicates the formation of multiple native-like anti-parallel beta-sheet conformations, kinetically trapped via subunit-association-induced hydrophobic collapse and stabilized by low-dielectric electrostatic interactions among/between Gly-28 and Lys-50 in opposing subunits. Results are discussed in terms of protein folding.

NMR structure of a de novo designed, peptide 33mer with two distinct, compact beta-sheet folds.

A de novo designed 33-residue polypeptide folds as a compact beta-sheet sandwich tetramer in aqueous solution. NMR structural analysis shows that although monomer subunits have the same three-stranded antiparallel beta-sheet fold, two equally populated conformational states are identified. Conformational heterogeneity arises from formation of two distinct dimer folds. Each dimer is formed by continuing the monomer beta-sheet into a six-stranded sheet similar to that found in alpha-chemokines. Dimer heterogeneity arises primarily from a two-residue shift in the alignment of interfacial strands. NOE-based conformational modeling has yielded well-defined structures for both dimer types. While the tetramer beta-sheet sandwich most probably results from association of hydrophobic surfaces from two amphipathic dimers, dimers could combine to form either two types of homotetramers and/or one heterotetramer composed of both dimer types. Even though interdimer NOEs could not be unambiguously identified to resolve this point, thermodynamic arguments based on observation of equal populations of both dimer types favor formation of heterotetramers.

Synthetic peptides probe folding initiation sites in platelet factor-4: stable chain reversal found within the hydrophobic sequence LIATLKNGRKISL.

Platelet factor-4 (PF4) is a 70-residue protein which contains a 3-stranded antiparallel beta-sheet domain on to which is folded a C-terminal alpha-helix and an aperiodic N-terminal region. In this study, three peptides derived from the beta-sheet (residues 24-46 and 38-57) and helix (residues 57-70) domains have been synthesized and studied in aqueous solution by CD and NMR. While peptides 24-46 and 56-70 demonstrate some weak conformational preferences, peptide 38-57 maintains a relatively well-defined, NOE-rich chain reversal sequence, L45-K46-N47-G48-R49-K50, which apparently is stabilized by hydrophobic side-chain interactions from the flanking sequences L41-L45 and I51-L53. Some helix-like conformational populations are noted in the native PF4 I42-A43-T44-L45 beta-strand segment. NOE-based distance geometry calculations yield native-like conformations within the L45-K50 sequence. Among 40 structures, backbone RMS deviations range from 0.5 to 1.2 A, and compared to the same sequence in native PF4, the average RMS deviation is 1.1 A. These results suggest that beta-sheet/turn residues L41-L53 present a folding initiation site on the PF4 folding pathway.

Effects of molecular association on structure and dynamics of a collagenous peptide.

Peptide GVKGDKGNPGWPGAPY from the triple-helix domain of type IV collagen aggregates in solution at a critical aggregation concentration of 18 mM. This molecular self-association process is investigated by 1H- and 13C-nmr spectroscopy. As a function of increasing peptide concentration, selective 1H resonances are cooperatively chemically shifted by up to 0.04 ppm to apparently saturable values at high concentration. Pulsed field gradient nmr was used to derive translation diffusion constants that, as the peptide concentration is increased, also cooperatively and monotonically decrease to an apparent limiting value. An average number of 6 monomer units per aggregate have been estimated from diffusion constant and 13C relaxation data. Comparative 1H nuclear Overhauser effect spectroscopy (NOESY) spectra accumulated at high and low peptide concentrations suggest that average internuclear distances are decreased as a result of peptide association. 13C-nmr multiple spin-lattice relaxation and 13C-[1H] NOE effects on 13C-enriched glycine methylene positions in the peptide demonstrate that overall molecular tumbling and backbone internal motions are attenuated in the aggregate state. Lowering the solution pD from pD 6 to pD 2 disrupts the aggregate state, suggesting a role for electrostatic interactions in the association process. Based on thermodynamic considerations, hydrophobic interactions also probably act to stabilize the aggregate state. These data are discussed in terms of an nmr/NOE constrained computer-modeled structure of the peptide.