Dynamics of Alpha-Helix Formation in the CSAW Model

We study the folding dynamics of polyalanine (Ala$_{20}$), a protein fragment with 20 residues whose native state is a single alpha helix. We use the CSAW model (conditioned self-avoiding walk), which treats the protein molecule as a chain in Brownian motion, with interactions that include hydrophobic forces and internal hydrogen bonding. We find that large scale structures form before small scale structures, and obtain the relevant relaxation times. We find that helix nucleation occurs at two separate points on the protein chain. The evolution of small and large scale structures involve different mechanisms. While the former can be describe by rate equations governing the growth of helical content, the latter is akin to the relaxation of an elastic solid.Comment: 18 pages, 10 figure

Alpha helix-coil phase transition: analysis of ab initio theory predictions

In the present paper we present results of calculations obtained with the use of the theoretical method described in our preceding paper [1] and perform detail analysis of alpha helix-random coil transition in alanine polypeptides of different length. We have calculated the potential energy surfaces of polypeptides with respect to their twisting degrees of freedom and construct a parameter-free partition function of the polypeptide using the suggested method [1]. From the build up partition function we derive various thermodynamical characteristics for alanine polypeptides of different length as a function of temperature. Thus, we analyze the temperature dependence of the heat capacity, latent heat and helicity for alanine polypeptides consisting of 21, 30, 40, 50 and 100 amino acids. Alternatively, we have obtained same thermodynamical characteristics from the use of molecular dynamics simulations and compared them with the results of the new statistical mechanics approach. The comparison proves the validity of the statistical mechanic approach and establishes its accuracy.Comment: 34 pages, 12 figure

Ab initio theory of helix-coil phase transition

In this paper we suggest a theoretical method based on the statistical mechanics for treating the alpha-helix-random coil transition in alanine polypeptides. We consider this process as a first-order phase transition and develop a theory which is free of model parameters and is based solely on fundamental physical principles. It describes essential thermodynamical properties of the system such as heat capacity, the phase transition temperature and others from the analysis of the polypeptide potential energy surface calculated as a function of two dihedral angles, responsible for the polypeptide twisting. The suggested theory is general and with some modification can be applied for the description of phase transitions in other complex molecular systems (e.g. proteins, DNA, nanotubes, atomic clusters, fullerenes).Comment: 24 pages, 3 figure

Investigating thermal stability based on the structural changes of lactase enzyme by several orthogonal methods

Thermal stability of lactase (β-galactosidase) enzyme has been studied by a variety of physico-chemical methods. β-galactosidase is the main active ingredient of medications for lactose intolerance. It is typically produced industrially by the Aspergillus oryzae filamentous fungus. Lactase was used as a model to help understand thermal stability of enzyme-type biopharmaceuticals. Enzyme activity (hydrolyzation of lactose) of β-galactosidase was determined after storing the solid enzyme substance at various temperatures. For a better understanding of the relationship between structure and activity changes we determined the mass and size of the molecules with gel electrophoresis and dynamic light scattering and detected aggregation processes. A bottom-up proteomic procedure was used to determine the primary amino acid sequence and to investigate changes in the N-glycosylation pattern of the protein. NMR and CD spectroscopic methods were used to observe changes in higher order structures and to reveal relationships between structural and functional changes

Thrombomodulin enhances complement regulation through strong affinity interactions with factor H and C3b-Factor H complex

Introduction Coagulation and complement systems are simultaneously activated at sites of tissue injury, leading to thrombin generation and opsonisation with C3b. Thrombomodulin (TM) is a cell-bound regulator of thrombin activation, but can also enhance the regulatory activity of complement factor H (FH), thus accelerating the degradation of C3b into inactive iC3b. Objectives This study sought to determine the biophysical interaction affinities of two recombinant TM analogs with thrombin, FH and C3b in order to analyze their ability to regulate serum complement activity. Methods Surface plasmon resonance (SPR) analysis was used to determine binding affinities of TM analogs with FH and C3b, and compared to thrombin as positive control. The capacity of the two recombinant TM analogs to regulate complement in serum was tested in standard complement hemolytic activity assays. Results SPR analysis showed that both TM analogs bind FH and C3b-Factor H with nanomolar and C3b with micromolar affinity; binding affinity for its natural ligand thrombin was several fold higher than for FH. At a physiological relevant concentration, TM inhibits complement hemolytic activity in serum via FH dependent and independent mechanisms. Conclusions TM exhibits significant binding affinity for complement protein FH and C3b-FH complex and its soluble form is capable at physiologically relevant concentrations of inhibiting complement activation in serum

Envelope Determinants of Equine Lentiviral Vaccine Protection

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for antibody binding, not neutralizing, assays that correlate with vaccine protection. © 2013 Craigo et al

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation

Allosteric Modulation of the HIV-1 gp120-gp41 Association Site by Adjacent gp120 Variable Region 1 (V1) N-Glycans Linked to Neutralization Sensitivity

The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/ or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and Cterminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn136 in V1 (T138N mutation) inconjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N139INN sequence, which ablates the overlapping Asn141-Asn142-Ser-Ser potential N-linked glycosylation sequons inV1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu593, Trp596 and Lys601. The 136 and/or 142glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection

Protein traffic is an intracellular target in alcohol toxicity

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains