Short Hydrophobic Sequence Research Articles

Exploring a Solvent Dependent Strategy to Control Self-Assembling Behavior and Cellular Interaction in Laminin-Mimetic Short Peptide based Supramolecular Hydrogels.

Self-assembled hydrogels, fabricated through diverse non-covalent interactions, have been extensively studied in regenerative medicines. Inspired from bioactive functional motifs of ECM protein, short peptide sequences have shown remarkable abilities to replicate the intrinsic features of the natural extracellular milieu. In this direction, we have fabricated two short hydrophobic bioactive sequences derived from the laminin protein i. e., IKVAV and YIGSR. Based on the substantial hydrophobicity of these peptides, we selected a co-solvent approach as a suitable gelation technique that included different concentrations of DMSO as an organic phase along with an aqueous solution containing 0.1 % TFA. These hydrophobic laminin-based bioactive peptides with limited solubility in aqueous physiological environment showed significantly enhanced solubility with higher DMSO content in water. The enhanced solubility resulted in extensive intermolecular interactions that led to the formation of hydrogels with a higher-order entangled network along with improved mechanical properties. Interestingly, by simply modulating DMSO content, highly tunable gels were accessed in the same gelator domain that displayed differential physicochemical properties. Further, the cellular studies substantiated the potential of these laminin-derived hydrogels in enhancing cell-matrix interactions, thereby reinforcing their applications in tissue engineering.

On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium.

In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of MreB biochemical and polymerization properties in vitro remains limited and is mostly based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized protofilaments by electron microscopy and the first 3D-structure of MreB from a Gram-positive bacterium. We show that Geobacillus stearothermophilus MreB forms straight pairs of protofilaments on lipid surfaces in the presence of ATP or GTP, but not in the presence of ADP, GDP or non-hydrolysable ATP analogs. We demonstrate that membrane anchoring is mediated by two spatially close short hydrophobic sequences while electrostatic interactions also contribute to lipid binding, and show that the population of membrane-bound protofilament doublets is in steady-state. In solution, protofilament doublets were not detected in any condition tested. Instead, MreB formed large sheets regardless of the bound nucleotide, albeit at a higher critical concentration. Altogether, our results indicate that both lipids and ATP are facilitators of MreB polymerization, and are consistent with a dual effect of ATP hydrolysis, in promoting both membrane binding and filaments assembly/disassembly.

Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation

ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

Investigating the Determinants of Substrate Binding Orientation within the E. coli Hsp70 Chaperone Protein

Molecular chaperone proteins play a critical role in maintenance of the cell’s proteome through protein quality control. Heat shock protein 70 chaperones (Hsp70s) are an evolutionarily conserved and diverse family of chaperone proteins that assist with proper protein folding and prevention of aggregation in the cell. Previous research has shown that the substrate‐binding domain of the E. coli Hsp70, DnaK, contains binding pockets of low stringency extending bilaterally from a highly hydrophobic central pocket. The SBD interacts with short hydrophobic sequences of substrates by “reading” the sequence. The SBD will then bind the substrate in one of two orientations: an N‐C, or forward, orientation, or a C‐N, reverse, orientation. Research done thus far on substrate binding has not revealed an exact mechanism that determines orientation. In this study, peptides possessing the same sequence with opposing backbone directions were used to test the significance of backbone interactions with the SBD as well as residue side chains and their interactions with the SBD. The analysis of the bound orientation of these peptides is accomplished by disulfide cross‐linking using peptides containing an N‐terminal Cys and SBD proteins with a Cys introduced in such a way where it can be reached in only one orientation. The results indicated slight energetic favorable biases for the interactions of residue side chains and the SBD pockets. The results also place a high importance on leucine residues in substrates as a determinant of optimal pocket ‐ residue fit and binding orientation. Altogether, the data suggest that binding occurs in the orientation that supports the most optimal fit of residue side chains into binding pockets, which reveals further intricacies and selectivity involved in substrate binding for Hsp70s.

Exploring Hsp70 chaperone interactions with a full‐length substrate

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding and preventing proteins from aggregation (Clérico et al., 2015). Despite extensive research that reveals a selective promiscuity between the Hsp70 substrate‐binding domain (SBD) and client proteins, there is a paucity of data on the interaction of Hsp70s with large protein clients. To investigate the interaction between Hsp70s and a full‐length substrate, we chose to characterize the binding of the E. coli homolog,DnaK, to the precursor of alkaline phosphatase (proPhoA). proPhoA is an endogenous substrate of DnaK that is unfolded and soluble under reducing conditions, and contains at least five sequences that bind DnaK when presented as short peptides (Rüdiger et al., 1997; Wild et al., 1992). To simplify the experimental conditions we used truncated versions of proPhoA (~100 residues long) that contain 1 or 2 binding sites. We used biophysical techniques to reveal the affinity of binding, and NMR spectroscopy to elucidate the mode of chaperone binding to the proPhoA fragments. We found that there is a hierarchy of affinities among the 5 potential binding sites, and novel cryptic binding sites have been observed. In addition, data suggest there may be a role for residues beyond the canonical five‐residue core sequences. The mechanism of DnaK binding site selection on a substrate containing multiple potential sites and the structural basis of interaction between the chaperone and its respective binding sites on a full‐length substrate will be described.Support or Funding InformationNIH R35 GM118161 MIRA Grant "Protein folding in the Cell: Challenges and Coping Mechanisms" 6/1/16 – 5/31/2021

Membrane Targeting and Insertion of the C-Tail Protein SciP

C-tailed membrane proteins insert into the bilayer post-translationally because the hydrophobic anchor segment leaves the ribosome at the end of translation. Nevertheless, we find here evidence that the targeting of SciP to the membrane of Escherichia coli occurs co-translationally since signal elements in the N-terminal part of the SciP protein sequence are present. Two short hydrophobic sequences were identified that targeted a green fluorescent protein-SciP fusion protein to the membrane involving the signal recognition particle. After targeting, the membrane insertion of SciP is catalyzed by YidC independent of the SecYEG translocase. However, when the C-terminal tail of SciP was extended to 21 aa residues, we found that SecYEG becomes involved and makes its membrane insertion more efficient.

A simple lattice model that captures protein folding, aggregation and amyloid formation.

The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to inter-molecular beta strand formation. Such amyloid formation is associated with neurodegenerative disorders like Alzheimer's and Parkinson's. Molecular modelling can play a key role in providing insight into the factors that make proteins prone to fibril formation. However, fully atomistic models are computationally too expensive to capture the length and time scales associated with fibril formation. As the ability to form fibrils is the rule rather than the exception, much insight can be gained from the study of coarse-grained models that capture the key generic features associated with amyloid formation. Here we present a simple lattice model that can capture both protein folding and beta strand formation. Unlike standard lattice models, this model explicitly incorporates the formation of hydrogen bonds and the directionality of side chains. The simplicity of our model makes it computationally feasible to investigate the interplay between folding, amorphous aggregation and fibril formation, and maintains the capability of classic lattice models to simulate protein folding with high specificity. In our model, the folded proteins contain structures that resemble naturally occurring beta-sheets, with alternating polar and hydrophobic amino acids. Moreover, fibrils with intermolecular cross-beta strand conformations can be formed spontaneously out of multiple short hydrophobic peptide sequences. Both the formation of hydrogen bonds in folded structures and in fibrils is strongly dependent on the amino acid sequence, indicating that hydrogen-bonding interactions alone are not strong enough to initiate the formation of beta sheets. This result agrees with experimental observations that beta sheet and amyloid formation is strongly sequence dependent, with hydrophobic sequences being more prone to form such structures. Our model should open the way to a systematic study of the interplay between the factors that lead to amyloid formation.

Subunit Organisation of In Vitro Reconstituted HOPS and CORVET Multisubunit Membrane Tethering Complexes

Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.

Aggregation gatekeepers modulate protein homeostasis of aggregating sequences and affect bacterial fitness

The most common mechanism by which proteins aggregate consists in the assembly of short hydrophobic primary sequence segments into extended β-structured agglomerates. A significant enrichment of charged residues is observed at the flank of these aggregation-prone sequence segments, suggesting selective pressure against aggregation. These so-called aggregation gatekeepers act by increasing the intrinsic solubility of aggregating sequences in vitro, but it has been suggested that they could also facilitate chaperone interactions. Here, we address whether aggregation gatekeepers affect bacterial fitness. In Escherichia coli MC4100 we overexpressed GFP fusions with an aggregation-prone segment of σ32 (further termed σ32β) flanked by gatekeeper and non-gatekeeper residues and measured pairwise competitive growth. We found that the identity of flanking residues had significant effect on bacterial growth. Overexpression of σ32β flanked by its natural gatekeepers displayed the greatest competitive fitness, followed by other combinations of gatekeepers, while absence of gatekeepers strongly affects bacterial fitness. Further analysis showed the diversity of effects of gatekeepers on the proteostasis of σ32β including synthesis and degradation rates, in vivo aggregation propensity and chaperone response. Our results suggest that gatekeeper residues affect bacterial fitness not only by modulating the intrinsic aggregation propensity of proteins but also by the manner in which they affect the processing of σ32β-GFP by the protein quality control machinery of the cell. In view of these observations, we hypothesize that variation at gatekeeper positions offers a flexible selective strategy to modulate the proteostatic regulation of proteins to the match intrinsic aggregation propensities of proteins with required expression levels.

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²⁺ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.

Characterization of Two β-1,3-Glucosyltransferases fromEscherichia coliSerotypes O56 and O152

The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-beta1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-alpha-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-beta1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid beta-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.

Flocculation of kaolin suspension with the adsorption of N,N-disubstituted hydrophobically modified polyacrylamide

The interaction between N,N-disubstituted hydrophobically modified polyacrylamide (HMPAM) and kaolin particle in aqueous suspension was investigated by determining the adsorption isotherms and the flocculation behaviors. HMPAMs with different structures varying in hydrophobic distribution degree, NH (the initial number of hydrophobic monomers per micelle during the preparation) 4.45, 2.55 and 1.79, were used in the experiment. Compared with polyacrylamide (PAM), the HMPAMs have very high-adsorbed amount that correspond to a thick adsorbed layer. These surface loads correlate positively with the effective flocculation of HMPAMs, which are attributed to the affinity and association behavior. Additionally, in the test, the HMPAMs with blocky hydrophobic distribution were better than the ones with short hydrophobic sequences. NaCl was used to detect the effect of electrolyte. As a result of the change of environment property with addition of NaCl, association behavior is promoted; consequently, adsorption and flocculation efficiency are enhanced.

Effect of Sequence Hydrophobicity and Bilayer Width upon the Minimum Length Required for the Formation of Transmembrane Helices in Membranes

The minimum hydrophobic length necessary to form a transmembrane (TM) helix in membranes was investigated using model membrane-inserted hydrophobic helices. The fluorescence of a Trp at the center of the sequence and its sensitivity to quenching were used to ascertain helix position within the membrane. Peptides with hydrophobic cores composed of poly(Leu) were compared to sequences containing a poly 1:1 Leu:Ala core (which have a hydrophobicity typical of natural TM helices). Studies varying bilayer width revealed that the poly(Leu) core peptides predominately formed a TM state when the bilayer width exceeded hydrophobic sequence length by (i.e. when negative mismatch was) up to ∼11-12 Å (e.g. the case of a 11-12 residue hydrophobic sequence in bilayers with a biologically relevant width, i.e. dioleoylphosphatidylcholine (DOPC) bilayers), while poly(LeuAla) core peptides formed predominantly TM state with negative mismatch of up to 9 Å (a 13 residue hydrophobic sequence in DOPC bilayers). This indicates that minimum length necessary to form a predominating amount of a TM state (minimum TM length) is only modestly hydrophobicity-dependent for the sequences studied here, and a formula that defines the minimum TM length as a function of hydrophobicity for moderately-to-highly hydrophobic sequences was derived. The minimum length able to form a stable TM helix for alternating LeuAla sequences, and that for sequences with a Leu block followed by an Ala block, was similar, suggesting that a hydrophobicity gradient along the sequence may not be an important factor in TM stability. TM stability was also similar for sequences flanked by different charged ionizable residues (Lys, His, Asp). However, ionizable flanking residues destabilized the TM configuration much more when charged than when uncharged. The ability of short hydrophobic sequences to form TM helices in membranes in the presence of substantial negative mismatch implies that lipid bilayers have a considerable ability to adjust to negative mismatch, and that short TM helices may be more common than generally believed. Factors that modulate the ability of bilayers to adjust to mismatch may strongly affect the configuration of short hydrophobic helices.

L-Ala Modified Analogues of Amyloid β-Peptide Residue 17-20: Self-Association and Amyloid-like Fibril Formation

l-Ala modified analogues of amyloid β-peptide residue 17-20 LVFF (-l-Leu-l-Val-l-Phe-l-Phe-) have been designed and synthesized to study their self-assembling propensity, the nature of intermolecular interactions and rationalize with short hydrophobic sequences in the middle of Aβ that have important role in the neuropathology of Alzheimer’s disease. The peptides sequences LVFA and LAFA have been adopted from the β-sheet region of non-amyloidogenic proteins (hemoglobin-like falvoprotein and ATP synthase C chain, respectively). All the reported peptides self-associate into amyloid-like fibrils which are readily stained with a physiological dye Congo red and exhibits green gold birefringence under polarized light. The solid state FTIR studies of the fibrils reveal that the reported peptides self-associate through intermolecular hydrogen bonds to form antiparallel β-sheet structure, which is also supported by molecular modeling studies. This result suggests that l-Ala analogous of Aβ17-20, LVFA and LAFA also have virtually identical aggregation behavior.

α-Aminoisobutyric acid modified protected analogues of β-amyloid residue 17–20: a change from sheet to helix

The strong intermolecular interactions mediated by short hydrophobic sequences (e.g., 17–20, -l-Leu-l-Val-l-Phe-l-Phe-) in the middle of Aβ are known to play a crucial role in the neuropathology of Alzheimer's disease. FTIR, TEM and Congo red binding studies indicated that a series of l-Ala substituted terminally protected peptides related to the sequence 17–20 of the β-amyloid peptide, adopted β-sheet conformations. However, the Aib-modified analogues disrupt the β-sheet structure and switch over to a 310 helix with increasing number of Aib residues. X-ray crystallography shed some light on the change from sheet to helix at atomic resolution.

Amino-Terminal Precursor Sequence Modulates Canine Distemper Virus Fusion Protein Function

The fusion (F) proteins of most paramyxoviruses are classical type I glycoproteins with a short hydrophobic leader sequence closely following the translation initiation codon. The predicted reading frame of the canine distemper virus (CDV) F protein is more complex, with a short hydrophobic sequence beginning 115 codons downstream of the first AUG. To verify if the sequence between the first AUG and the hydrophobic region is translated, we produced a specific antiserum that indeed detected a short-lived F protein precursor that we named PreF(0). A peptide resulting from PreF(0) cleavage was identified and named Pre, and its half-life was measured to be about 30 min. PreF(0) cleavage was completed before proteolytic activation of F(0) into its F(1) and F(2) subunits by furin. To test the hypothesis that the Pre peptide may influence protein activity, we compared the function of F proteins synthesized with that peptide to that of F proteins synthesized with a shorter amino-terminal signal sequence. F proteins synthesized with the Pre peptide were more stable and less active. Thus, the Pre peptide modulates the function of the CDV F protein. Interestingly, a distinct two-hit activation process has been recently described for human respiratory syncytial virus, another paramyxovirus.

Starvation Promotes Nuclear Accumulation of the hsp70 Ssa4p in Yeast Cells

Nuclear import of proteins that are too large to passively enter the nucleus requires soluble factors, energy, and a nuclear localization signal (NLS). Nuclear protein transport can be regulated, and different forms of stress affect nucleocytoplasmic trafficking. As such, import of proteins containing a classical NLS is inhibited in starving yeast cells. In contrast, the hsp70 Ssa4p concentrates in nuclei upon starvation. Nuclear concentration of Ssa4p in starving cells is reversible, and transfer of stationary phase cells to fresh medium induces Ssa4p nuclear export. This export reaction represents an active process that is sensitive to oxidative stress. In starving cells, the N-terminal domain of Ssa4p mediates Ssa4p nuclear accumulation, and a short hydrophobic sequence, termed Star (for starvation), is sufficient to localize the reporter proteins green fluorescent protein or beta-galactosidase to nuclei. To determine whether nuclear accumulation of Star-beta-galactosidase depends on a specific nuclear carrier, we have analyzed its distribution in mutant yeast strains that carry a deletion of a single beta-importin gene. With this assay we have identified Nmd5p as a beta-importin required to concentrate Star-beta-galactosidase in nuclei when cells enter stationary phase.

A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human ?-lactalbumin

The molten globule state of alpha-lactalbumin (alpha LA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3(10) helices (residues 101-120) of alpha LA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of alpha LA make to the structure and stability of the alpha LA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.

Genetic Dissection of hTAFII130 Defines a Hydrophobic Surface Required for Interaction with Glutamine-rich Activators

The general transcription factor TFIID is a multiprotein complex consisting of the TATA box-binding protein and multiple TATA box-binding protein-associated factors (TAF(II)s). The central domain of human TAF(II)130 contains four glutamine-rich regions Q1-Q4 that interact with transcriptional activators such as Sp1 and CREB and mediate activation. We screened in yeast random point mutations introduced into Q1-Q4 against the Sp1 activation domain and obtained a distinct set of hTAF(II)130s with alterations in TAF(II)-activator interaction. Here we characterize functionally an hTAF(II)130 mutant containing a phenylalanine to serine change at position 311 (F311S) that is compromised in its ability to associate with Sp1B and CREB-N activation domains. Substitution of phenylalanine with tyrosine but not with isoleucine or tryptophan also reduced hTAF(II)130 interaction, suggesting that the hydrophobic character rather than the specific amino acid at this position is a key determinant of interaction. Deletion of nine amino acids (Delta9) surrounding Phe(311) abolished the interaction of hTAF(II)130 with Sp1. Overexpression of hTAF(II)130Q1/Q2 and Q1-Q4 strongly inhibited Sp1-dependent transcriptional enhancement in transient transfection assays, whereas expression of either F311S or Delta9 only partially suppressed Sp1-mediated activation. Thus, a short hydrophobic sequence motif encompassing Phe(311) in hTAF(II)130 represents a critical surface with which Sp1B interacts to activate transcription.

Activator-sigma interaction: a hydrophobic segment mediates the interaction of a sigma family promoter recognition protein with a sliding clamp transcription activator

Activation of transcription at bacteriophage T4 late promoters and coupling of late transcription to concurrent replication requires a peculiar transcriptional activator, the gp45 sliding clamp of the T4 DNA polymerase. In order to activate transcription, the topologically DNA-linked trimeric gp45 must interact with two T4-encoded RNA polymerase-binding proteins, the gp33 co-activator, and the gp55 late sigma factor. The carboxy termini of gp55 and gp33 share a similar sequence, which has been shown to be required for response of late transcription to activation by gp45. Alanine-scanning mutagenesis of the C terminus of gp55 shows that residues within the short hydrophobic sequence L(D/A)FLYE, are necessary for gp55 to bind to gp45, and to respond maximally to transcriptional activation by gp45. When fused to GST, the peptide SLDFLYE suffices for specific gp45 binding. Thus, it constitutes the main gp55 epitope for gp45 interaction.